Marker for early diagnosis of kidney failure

ABSTRACT

The present invention addresses the problem of identifying a biomarker of renal failure, said biomarker being available from urine or blood, and fluctuating from an early stage than glomerular filtration rate and serum creatinine level, and thus developing a technique for diagnosing early stage kidney failure. A method for analyzing the blood, plasma, serum or urine of a renal failure suspected subject comprises a step of measuring the concentration of a pair of D-form and L-form of at least one amino acid selected from the amino acid group consisting of [D-serine] and [L-serine], etc., contained in the blood, plasma, serum or urine of the subject, and calculating, as an pathological index of the subject, the ratio of the D-form concentration to the L-form concentration or the percentage of the D-form concentration relative to the total concentration of the D-form and L-form.

TECHNICAL FIELD

The present invention relates to a method for analyzing the blood or urine of a subject suspected of renal failure, and a system for analyzing a blood or urine sample of a subject suspected of renal failure. More particularly, the present invention relates to a method for analyzing the blood, plasma, serum or urine of a subject that comprises a step for measuring the concentration of a pair of D-form and L-form of at least one amino acid selected from the group consisting of [D-serine] and [L-serine] and the like in the blood, plasma, serum or urine of the subject, and to a system for analyzing a blood, plasma, serum or urine sample of the subject for performing the aforementioned analysis method, comprising a memory unit, an analysis and measurement unit, a data processing unit and a pathological information output unit.

BACKGROUND ART

Chronic kidney disease (CKD) is a disease that affects 13.3 million Japanese, corresponding to roughly 13% of the Japanese adult population, and threatens the health of Japanese citizens due to the risk of progressing to end stage kidney disease (ESKD). Chronic kidney disease includes all pathological states in which depressed renal function as represented by glomerular filtration rate is found, or findings suggesting kidney damage persist in a chronic state (three months or longer). There is no effective treatment method for chronic kidney disease, and if chronic kidney disease progresses resulting in further depression of renal function, there is the risk of uremia, resulting in the need for artificial dialysis or kidney transplant, which will place a considerable burden on the patient in terms of health care costs (Non-Patent Document 1). Chronic kidney disease does not exhibit subjective symptoms. Diagnosis using markers for early diagnosis of renal failure is necessary for early diagnosis of chronic kidney disease and inhibition of its progression. However, there is currently no biomarker that is satisfactory in terms of accurately reflecting the progression of renal dysfunction at an earlier stage than the occurrence of changes in renal function as represented by glomerular filtration rate.

An experimental animal model of acute kidney injury (AKI) is able to reproduce the early stages of renal dysfunction. Acute kidney injury is a disease in which renal function decreases rapidly over several weeks or several days. A known model of this disease is an acute kidney injury experimental model which is induced by a surgical procedure or administration of a drug. The “gold standards” for diagnosing acute kidney injury are urine production volume and serum creatinine concentration. Serum creatinine concentration is superior in that it can be evaluated without performing a biopsy regardless of the presence or absence of urination. However, glomerular filtration rate is required to be in a steady state. In an experimental animal model of acute kidney injury, which is not sensitive to small fluctuations in glomerular filtration rate, changes in glomerular filtration rate become apparent at a comparatively late stage. Since serum creatinine concentration also fluctuates due to conditions such as age, gender, muscle mass or medications being taken at the time, it cannot be a specific marker (Non-Patent Document 2). Reported examples of markers for acute kidney injury include neutrophil gelatinase-associated lipocalin (NGAL), interleukin-18 (IL-18), kidney injury molecule-1 (KIM-1), proteins such as fatty acid binding proteins or cystatin C, and metabolic low molecular weight compounds such as homovanillic acid sulfate or trimethylamine-N-oxide. However, none of these markers are detected in the early stage of renal failure.

Since concentrations of D-serine and D-alanine in the serum of renal failure patients are higher than serum concentrations in normal individuals, and both the D-form concentrations and ratio of D-form concentration/(D-form concentration+L-form concentration) correlate with creatinine, these amino acids have been suggested to be candidates for markers of renal proximal tubular dysfunction (Non-Patent Document 3). It is also disclosed that D-amino acids (Ala, Pro, Ser) in the serum of nephritis patients tend to be elevated and have a correlation with creatinine level (Non-Patent Document 9). In addition, D-alanine, D-serine, D-glutamic acid and D-aspartic acid are observed in the serum of renal failure patients, and because of this, measurement of serum D-alanine concentration has been suggested to be useful in the diagnosis of renal failure (Non-Patent Document 5). D-serine and D-alanine concentration in the urine along with the ratio of the D-form to the total of the D-form and L-form were investigated in healthy individuals of various age groups, and it was suggested that processing of D-serine in the kidneys is different (Non-Patent Document 6). Although one or more amino acids selected from the group consisting of D-serine, D-threonine, D-alanine, D-asparagine, D-allo-threonine, D-glutamine, D-proline and D-phenylalanine were disclosed as being able to be used as pathological indicators of kidney disease (Patent Document 1). However, even though it is described in this document that a body fluid such as blood, plasma or urine is used as a specimen, blood is the only specimen used to determine pathological indicators for kidney disease in the examples, while there is nothing disclosed as to whether or not amino acids present in urine can be used as pathological indicators of kidney disease. It is also disclosed that the D-form ratios of alanine, valine, proline, threonine, aspartic acid and asparagine increase significantly in the urine of renal failure patients, while there is no significant differences with respect to methionine and serine (Non-Patent Document 7). However, in this document, although the ratio of the D-form concentration/(D-form concentration+L-form concentration) of each amino acid is calculated in renal failure patients for which pathological assessment criteria have not been indicated, increases in these ratios are merely indicated randomly (or vaguely) irrespective of disease stage, and there are no descriptions or suggestions as to the fact that fluctuations in these ratios correlate with pathological or other biomarkers of renal failure. After the priority date of the present application, urinary D/L-serine ratio was suggested to be able to be used as a biomarker capable of detecting early stage ischemic renal failure and classifying pathological stage since it decreases over time following ischemia reperfusion injury (Non-Patent Document 8). D-amino acid oxidase, which is involved in the decomposition of D-amino acids, is expressed in renal proximal tubules, and the enzyme activity of D-amino acid oxidase is known to decrease in ischemia reperfusion model rats (Non-Patent Document 4). L-serine is reabsorbed while D-serine is hardly reabsorbed at all under physiological conditions. However, the manner in which D-serine and other D-amino acids fluctuate in the early stage of renal failure is unknown.

PRIOR ART DOCUMENTS Patent Documents

-   Patent Document 1: International Publication No. WO 2013/140785

Non-Patent Documents

-   Non-Patent Document 1: KDIGO 2012, Clinical Practice Guideline for     the Evaluation and Management of Chronic Kidney Disease, Kidney     International Supplements 1 (2013) -   Non-Patent Document 2: Slocum, J. L., et al, Transl. Res. 159: 277     (2012) -   Non-Patent Document 3: Fukushima, T., et al, Biol. Pharm. Bull. 18:     1130 (1995) -   Non-Patent Document 4: Zhang, H., et al, Amino Acids 42: 337 (2012) -   Non-Patent Document 5: Ishida, et al, Kitasato Medical Journal, 23:     51-62 (1993) -   Non-Patent Document 6: Yong Huang, et al, Biol. Pharm. Bull. 21:     (2)156-162 (1998) -   Non-Patent Document 7: Magdalena C. Waldhier et al, Chromatography     B (2010) 1103-1112 -   Non-Patent Document 8: Jumpeji Sasabe et al, PLOS ONE, Vol. 9, Issue     1, e86504 -   Non-Patent Document 9: Nagata, Y., Viva Origino Vol. 18 (No. 2)     (1990), 15th Academic Symposium, Collection of Abstracts

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

There is a need to develop a technology for diagnosis of early stage renal failure by identifying a biomarker of renal failure that can be collected from urine or blood and fluctuates at an earlier stage than glomerular filtration rate and serum creatinine concentration.

Means for Solving the Problems

The present invention provides a method for analyzing the blood or urine of a subject suspected of renal failure. The analysis method of the present invention comprises a step for measuring the concentration of a pair of D-form and L-form of at least one amino acid selected from the amino acid group consisting of [D-serine] and [L-serine], [D-histidine] and [L-histidine], [D-asparagine] and [L-asparagine], [D-arginine] and [L-arginine], [D-allo-threonine] and [L-threonine], [D-glutamic acid] and [L-glutamic acid], [D-alanine] and [L-alanine], [D-proline] and [L-proline], [D-valine] and [L-valine], [D-allo-isoleucine] and [L-isoleucine], [D-phenylalanine] and [L-phenylalanine], and [D-lysine] and [L-lysine], and a step for calculating a pathological index value from the concentration of a pair of D-form and L-form of the at least one type of amino acid. Here, the pathological index value calculated from a pair of D-form and L-form amino acid enables to correlate a subject with the pathology of kidney disease in the case where a significant decrease in the proportion of the D-form is indicated. For example, the ratio of the concentration of the D-form to the concentration of the L-form of at least one amino acid, or the ratio or percentage of the concentration of the D-form to the sum of the concentrations of the D-form and the L-form, can be calculated as the pathological index value of the subject. Although amino acids in the urine of renal failure patients whose pathological assessment criteria have not been indicated are analyzed in Non-Patent Document 7, it is disclosed that the D-amino acid ratio for alanine, valine, proline, threonine and aspartic acid is significantly increased. Thus, a decrease in the proportion of D-amino acids in the urine of renal failure patients is a surprising finding.

In another mode of the analysis method of the present invention, [D-glutamine] and [L-glutamine], [D-threonine] and [L-threonine], [D-allo-threonine] and [L-allo-threonine] and [D-leucine] and [L-leucine] may be included in the aforementioned amino acid group. Thus, the analysis method of the present invention may comprise a step for measuring the concentration of a pair of D-form and L-form of at least one amino acid selected from the amino acid group consisting of [D-serine] and [L-serine], [D-histidine] and [L-histidine], [D-asparagine] and [L-asparagine], [D-arginine] and [L-arginine], [D-allo-threonine] and [L-threonine], [D-glutamic acid] and [L-glutamic acid], [D-alanine] and [L-alanine], [D-proline] and [L-proline], [D-valine] and [L-valine], [D-allo-isoleucine] and [L-isoleucine], [D-phenylalanine] and [L-phenylalanine], [D-lysine] and [L-lysine], [D-glutamine] and [L-glutamine], [D-threonine] and [L-threonine], [D-allo-threonine] and [L-allo-threonine], and [D-leucine] and [L-leucine], and a step for calculating a pathological index value from the concentration of a pair of D-form and L-form of the at least one amino acid. Here, the pathological index value calculated from a pair of D-form and L-form of at least one amino acid enables to correlate a subject with the pathology of kidney disease, when a significant decrease in the proportion of the D-form is indicated. For example, the ratio of the concentration of the D-form to the concentration of the L-form of at least one amino acids, or the ratio or percentage of the concentration of the D-form to the sum of the concentrations of the D-form and the L-form, can be calculated as the pathological index value of the subject.

Another mode of the present invention may also relate to a detection method for detecting renal failure, comprising a step for measuring the concentration of a pair of D-form and L-form of at least one amino acid selected from the amino acid group consisting of [D-serine] and [L-serine], [D-histidine] and [L-histidine], [D-asparagine] and [L-asparagine], [D-arginine] and [L-arginine], [D-allo-threonine] and [L-threonine], [D-glutamic acid] and [L-glutamic acid], [D-alanine] and [L-alanine], [D-proline] and [L-proline], [D-valine] and [L-valine], [D-allo-isoleucine] and [L-isoleucine], [D-phenylalanine] and [L-phenylalanine], [D-lysine] and [L-lysine], [D-glutamine] and [L-glutamine], [D-threonine] and [L-threonine], [D-allo-threonine] and [L-allo-threonine], and [D-leucine] and [L-leucine] in the urine of a subject, and

a step for calculating a pathological index value from the concentration of a pair of D-form and L-form of at least one amino acid that correlates a decrease in the proportion of a D-form with renal failure of the subject.

Here, the detection method relates to a method that may be performed by a non-physician such as a medical assistant or may be performed by an analysis facility.

Although an amino acid may be arbitrarily selected from the aforementioned amino acid group, it is more preferably selected from the group consisting of [D-histidine] and [L-histidine], [D-arginine] and [L-arginine], [D-glutamic acid] and [L-glutamic acid], [D-valine] and [L-valine], [D-allo-isoleucine] and [L-isoleucine], [D-lysine] and [L-lysine], [D-glutamine] and [L-glutamine], [D-leucine] and [L-leucine], and [D-allo-threonine] and [L-allo-threonine]. In another mode, each combination of amino acids may be excluded from the aforementioned amino acid group. For example, [D-serine] and [L-serine] may be excluded, [D-histidine] and [L-histidine] may be excluded, [D-asparagine] and [L-asparagine] may be excluded, [D-arginine] and [L-arginine]may be excluded, [D-allo-threonine] and [L-threonine] may be excluded, [D-glutamic acid] and [L-glutamic acid] may be excluded, [D-alanine] and [L-alanine] may be excluded, [L-proline] and [L-proline] may be excluded, [D-valine] and [L-valine] may be excluded, [D-allo-isoleucine] and [L-isoleucine] may be excluded, [D-phenylalanine] and [L-phenylalanine] may be excluded, [D-lysine] and [L-lysine] may be excluded, [D-glutamine] and [L-glutamine] may be excluded, [D-threonine] and [L-threonine] may be excluded, [D-allo-threonine] and [L-allo-w threonine] may be excluded, and [D-leucine] and [L-leucine] may be excluded.

These pathological index values have been indicated to decrease following renal ischemia reperfusion in the same manner as urine creatinine concentration. Since a decrease in urine creatinine concentration indicates depression of renal function, it can be used as a marker of renal failure, and a pathological index value of the present application can also be used as a marker of renal failure in the same manner as urine creatinine concentration. Furthermore, urine renal failure markers in the form of KIM-1 and NGAL demonstrate increases in urine concentration in response to depressed renal function. In an experimental model of ischemia reperfusion, although urine creatinine concentration decreased significantly 8 hours after ischemia reperfusion, urine KIM-1 increases significantly 20 hours after ischemia reperfusion and urine NGAL increased significantly 8 hours after ischemia reperfusion (FIGS. 2-D, 2-F and 2-G). Since a pathological index value calculated using the concentration of a pair of D-form and L-form of one or more amino acids selected from the group consisting of [D-serine] and [L-serine], [D-histidine] and [L-histidine], [D-allo-threonine] and [L-allo-threonine], [D-asparagine] and [L-asparagine], [D-arginine] and [L-arginine], [D-alanine] and [L-alanine], [D-proline] and [L-proline], and [D-lysine] and [L-lysine] decreases significantly at a level of significance of P<0.05 4 hours after renal ischemia reperfusion, it can be said to have higher sensitivity for renal failure. Thus, this pathological index value is preferably used from the viewpoint of having higher sensitivity for early stage renal failure. More preferably, since a pathological index value calculated using the concentration of a pair of D-form and L-form of one or more amino acids selected from the group consisting of [D-histidine] and [L-histidine], [D-asparagine] and [L-asparagine], [D-proline] and [L-proline], and [D-lysine] and [L-lysine] decreases significantly at a level of significance of P<0.01 4 hours after renal ischemia reperfusion, this pathological index value can be used as a marker having higher sensitivity. Even more preferably, since a pathological index value calculated from concentration of a pair of D-form and L-form of one or more amino acids selected from the group consisting of [D-histidine] and [L-histidine], [D-proline] and [L-proline], and [D-lysine] and [L-lysine] decreases significantly at a level of significance of P<0.001 4 hours after renal ischemia reperfusion, this pathological index value can be used as a marker having extremely high sensitivity. Thus, the pathological index value of the present invention has sensitivity that is equal to or higher than that of conventional renal failure markers such as serum creatinine, and can be used for early diagnosis of renal failure. Examples of early stage renal failure include a state in which serum creatinine has increased by 1.5 to 2.0 times, which is the criterion for the early stage of each of the RIFLE, AKIN and KDIGO classifications of acute kidney injury (AKI), a state wherein renal function reduction in chronic kidney disease (CKD) indicated by GFR is started, or an extremely early state prior to the appearance of changes in serum creatinine or GFR even though renal failure is present. In addition, a pathological index value calculated using the concentration of a pair of D-form and L-form of one or more amino acids selected from the group consisting of [D-glutamic acid] and [L-glutamic acid], [D-phenylalanine] and [L-phenylalanine], [D-valine] and [L-valine], [D-glutamine] and [L-glutamine], [D-threonine] and [L-threonine], [D-leucine] and [L-leucine], [D-allo-isoleucine] and [L-isoleucine], and [D-allo-threonine] and [L-threonine], which do not exhibit a significant difference 4 hours after renal ischemia reperfusion but exhibit a significant difference 8 hours after renal ischemia reperfusion, can be used as a marker having sensitivity equal to that of conventional markers such as creatinine.

The pathological index value calculated from the concentration of D-form and L-form of one amino acid may be used, or the pathological index value calculated from the concentration of another D-form and L-form of another amino acid can be used in combination therewith. For example, a pathological index value having extremely high sensitivity calculated from the concentration of D-form and L-form of one or more amino acids selected from the group consisting of [D-histidine] and [L-histidine], [D-proline] and [L-proline], and [D-lysine] and [L-lysine] may be combined with another pathological index value having extremely high sensitivity calculated from the concentration of a D-form and L-form of an amino acid selected from the same group. In addition, in a different mode, the aforementioned pathological index value having extremely high sensitivity can be used in combination with a pathological index value having a lower sensitivity, for example, a pathological index value calculated from the concentration of a D-form and L-form of one amino acid selected from the group consisting of [D-glutamic acid] and [L-glutamic acid], [D-phenylalanine] and [L-phenylalanine], [D-valine] and [L-valine], [D-glutamine] and [L-glutamine], [D-threonine] and [L-threonine], [D-leucine] and [L-leucine], [D-allo-isoleucine] and [L-isoleucine], and [D-allo-threonine] and [L-threonine], which enables sensitively detecting kidney injury in comparison with urine creatinine, urine KIM-1 or urine NGAL in a state in which depressed renal function, in which serum creatinine has increased by a factor of 1.5 to 2.0, has occurred or a state prior thereto.

The analysis method of the present invention may further comprise a step for determining whether the pathological index value of a subject is similar to the pathological index reference value of a healthy individual, whether the pathological index value of a subject is similar to the pathological index reference value of an acute renal failure patient or chronic renal failure patient, or whether the pathological index value of a subject is between the pathological index reference value of a healthy individual and the pathological index reference value of a chronic renal failure patient, by comparing the pathological index value of the subject with the pathological index reference value of a healthy individual and the pathological index reference value of an acute renal failure patient and/or chronic renal failure patient.

In still another mode, the present invention makes it possible to determine whether a subject suffers renal failure by preliminarily setting a threshold value based on the pathological index values of a healthy individual group and/or renal failure patient group, and then comparing the pathological index value of a subject with the threshold value. A person with ordinary skill in the art is able to suitably set a threshold value from the pathological index values of a healthy individual group and renal failure patient group. Although the average value, median value or X percentile value of the healthy individual group or renal failure patient group can be used for the threshold value, the threshold value is not limited thereto. Here, an arbitrary numerical value can be selected for X, and a value of 3, 5, 10, 15, 20, 30, 40, 60, 70, 80, 85, 90, 95 or 97 can be suitably used. Only one threshold value may be used or a plurality of threshold values can be set depending on the type of renal failure (acute or chronic), the cause thereof (such as drug-induced nephropathy, diabetic nephropathy, IgA nephropathy, membranous nephropathy or nephrosclerosis), disease state (early, intermediate or late) and the amino acids or combination thereof used. The pathology of the renal failure of a subject can be assessed, determined or diagnosed by comparing the pathological index value of the subject with a preset threshold value.

The analysis method of the present invention may further comprise a step for determining whether a first pathological index value of a subject, calculated from the concentration of a pair of D-form and L-form of at least one amino acid selected from the amino acid group consisting of [D-serine] and [L-serine], [D-histidine] and [L-histidine], [D-asparagine] and [L-asparagine], [D-arginine] and [L-arginine], [D-allo-threonine] and [L-threonine], [D-alanine] and [L-alanine], [D-proline] and [L-proline], [D-valine] and [L-valine], and [D-lysine] and [L-lysine] in the blood, plasma, serum or urine of the subject, is between the pathological index reference value of a healthy individual and the pathological index reference value of a chronic renal failure patient, and whether the second pathological index value of the subject, calculated from the concentration of a pair of D-form and L-form of at least one amino acid among [D-glutamic] and [L-glutamic acid], [D-allo-isoleucine] and [L-isoleucine] and [D-phenylalanine] and [L-phenylalanine] in the blood, plasma, serum or urine of the subject, is between a pathological index reference value of the healthy individual and the pathological index reference value of the chronic renal failure patient.

In the present invention, the pathological index value of a subject calculated from the concentration of a pair of D-form and L-form of at least one amino acid selected from the group consisting of [D-serine] and [L-serine], [D-histidine] and [L-histidine], [D-asparagine] and [L-asparagine], [D-arginine] and [L-arginine], [D-alanine] and [L-alanine], [D-proline] and [L-proline], and [D-lysine] and [L-lysine] can be used as a first pathological index value, while the pathological index value of the subject calculated from the concentration of a pair of D-form and L-form of at least one amino acid selected from the group consisting of [D-glutamic acid] and [L-glutamic acid], [D-allo-isoleucine] and [L-isoleucine], [D-phenylalanine] and [L-phenylalanine], [D-valine] and [L-valine], and [D-allo-threonine] and [L-threonine] can be used as a second pathological index value.

In the case [D-glutamine] and [L-glutamine], [D-threonine] and [L-threonine], [D-allo-threonine] and [L-allo-threonine] and [D-leucine] and [L-leucine] are included in the aforementioned amino acid group, the pathological index value of the subject calculated from the concentration of a pair of D-form and L-form of at least one amino acid selected from the group consisting of [D-serine] and [L-serine], [D-histidine] and [L-histidine], [D-asparagine] and [L-asparagine], [D-arginine] and [L-arginine], [D-alanine] and [L-alanine], [D-proline] and [L-proline], [D-lysine] and [L-lysine], and [D-allo-threonine] and [L-allo-threonine] can be used for the first pathological index value. On the other hand, the pathological index value calculated from the concentration of a pair of D-form and L-form of at least one amino acid selected from the group consisting of [D-glutamine] and [L-glutamine], [D-threonine] and [L-threonine], and [D-leucine] and [L-leucine] in addition to [D-glutamic acid] and [L-glutamic acid], [D-allo-isoleucine] and [L-isoleucine], [D-phenylalanine] and [L-phenylalanine], and [D-valine and L-valine] can be used for the second pathological index value.

The present invention provides a system for analyzing a blood or urine sample of a subject suspected of renal failure. The sample analysis system of the present invention comprises a memory unit, an analysis and measurement unit, a data processing unit and a pathological information output unit. The aforementioned memory unit stores the pathological index reference values for the blood, plasma, serum or urine of healthy individuals, and the pathological index reference values for the blood, plasma, serum or urine of acute renal failure patients and/or chronic renal failure patients. The aforementioned analysis and measurement unit separates and quantifies at least one pair of amino acid stereoisomers present in the blood, plasma, serum or urine of the aforementioned subject selected from the group consisting of D-serine and L-serine, D-histidine and L-histidine, D-asparagine and L-asparagine, D-arginine and L-arginine, D-allo-threonine and L-threonine, D-glutamic acid and L-glutamic acid, D-alanine and L-alanine, D-proline and L-proline, D-valine and L-valine, D-allo-isoleucine and L-isoleucine, D-phenylalanine and L-phenylalanine, and D-lysine and L-lysine. The aforementioned data processing unit performs a step for calculating the pathological index value of the aforementioned subject from the concentration of a pair of D-form and L-form of at least one amino acid of the group consisting of [D-serine] and [L-serine], [D-histidine] and [L-histidine], [D-asparagine] and [L-asparagine], [D-arginine] and [L-arginine], [D-allo-threonine] and [L-threonine], [D-glutamic acid] and [L-glutamic acid], [D-alanine] and [L-alanine], [D-proline] and [L-proline], [D-valine] and [L-valine], [D-allo-isoleucine] and [L-isoleucine], [D-phenylalanine] and [L-phenylalanine], and [D-lysine] and [L-lysine]. Here, the pathological index value calculated from the concentration of a pair of D-form and L-form of at least one amino acid enables a subject to be correlated with renal failure in the case of indicating a decrease in the composition ratio of the D-form.

In a different mode, D-glutamine and L-glutamine], D-threonine and L-threonine], [D-allo-threonine and L-allo-threonine and D-leucine and L-leucine may be included in addition to the aforementioned amino acid pairs. Thus, in a different mode, the aforementioned analysis and measurement unit separates and quantifies at least one pair of amino acid stereoisomers present in the blood, plasma, serum or urine of the aforementioned subject, selected from the group consisting of D-serine and L-serine, D-histidine and L-histidine, D-asparagine and L-asparagine, D-arginine and L-arginine, D-allo-threonine and L-threonine, D-glutamic acid and L-glutamic acid, D-alanine and L-alanine, D-proline and L-proline, D-valine and L-valine, D-allo-isoleucine and L-isoleucine, D-phenylalanine and L-phenylalanine, D-lysine and L-lysine, D-glutamine and L-glutamine, D-threonine and L-threonine, D-allo-threonine and L-allo-threonine and D-leucine and L-leucine. The aforementioned data processing unit performs a step for calculating the pathological index value of the aforementioned subject from the concentration of a pair of D-form and L-form of at least one amino acid selected from the group consisting of [D-serine] and [L-serine], [D-histidine] and [L-histidine], [D-asparagine] and [L-asparagine], [D-arginine] and [L-arginine], [D-allo-threonine] and [L-threonine], [D-glutamic acid] and [L-glutamic acid], [D-alanine] and [L-alanine], [D-proline] and [L-proline], [D-valine] and [L-valine], [D-allo-isoleucine] and [L-isoleucine], [D-phenylalanine] and [L-phenylalanine], [D-lysine] and [L-lysine], [D-glutamine] and [L-glutamine], [D-threonine] and [L-threonine], [D-allo-threonine] and [L-allo-threonine] and [D-leucine] and [L-leucine]. Here, the pathological index value calculated from the concentration of a pair of D-form and L-form of at least one amino acid enables a subject to be correlated with renal failure in the case of indicating a decrease in the composition ratio of the D-form.

The pathological index value refers to a value that can be calculated from the concentration of a certain D-form and L-form of a certain amino acid, and enables a subject to be correlated with renal failure in the case of indicating a decrease in the composition ratio of the D-form. In the sample analysis system of the present invention, for example, in relation to a D-form and L-form pair, the ratio of the concentration of the D-form to the concentration of the L-form, and the ratio or percentage of the concentration of a D-form to the sum of the concentrations of the D-form and L-form, is defined as the pathological index value of a subject. When the pathological index value of the subject is compared with the pathological index reference value of a healthy individual and the pathological index reference value of a patient with acute renal failure and/or chronic renal failure, and the pathological index value of the subject is similar to the pathological index reference value of the healthy individual, information that the aforementioned subject is hardly suspected of renal failure is defined as pathological information of the aforementioned subject. When the pathological index value of the aforementioned subject is similar to the pathological index reference value of the aforementioned chronic renal failure patient, information that the aforementioned subject is suspected of renal failure is defined as pathological information of the aforementioned subject. When the pathological index value of the aforementioned subject is between the pathological index reference value of the aforementioned healthy individual and the pathological index reference value of the aforementioned chronic renal failure patient, information that the aforementioned subject is suspected of early stage renal failure is defined as pathological information of the aforementioned subject. The aforementioned pathological information output unit outputs the pathological information of the aforementioned subject.

In the sample analysis system of the present invention, when a first pathological index value of a subject calculated from the concentration in the blood, plasma, serum or urine of the aforementioned subject of a pair of D-form and L-form of at least one amino acid selected from the group consisting of [D-serine] and [L-serine], [D-histidine] and [L-histidine], [D-asparagine] and [L-asparagine], [D-arginine] and [L-arginine], [D-allo-threonine] and [L-threonine], [D-glutamic acid] and [L-glutamic acid], [D-alanine] and [L-alanine], [D-proline] and [L-proline], [D-valine] and [L-valine], [D-allo-isoleucine] and [L-isoleucine], [D-phenylalanine] and [L-phenylalanine], and [D-lysine] and [L-lysine] is between the pathological index reference value of the aforementioned healthy individual and the pathological index reference value of the aforementioned chronic renal failure patient, and a second pathological index value of the aforementioned subject calculated from the concentration in the blood, plasma, serum or urine of the aforementioned subject of a pair of D-form and L-form of at least one amino acid selected from the group consisting of [D-glutamic acid] and [L-glutamic acid], [D-allo-isoleucine] and [L-isoleucine], and [D-phenylalanine] and [L-phenylalanine] is between the pathological index reference value of the aforementioned healthy individual and the pathological index reference value of the aforementioned chronic renal failure patient, information that the aforementioned subject is suspected of extremely early stage renal failure is defined as pathological information of the aforementioned subject.

The present invention provides a method for diagnosing renal failure. The diagnostic method of the present invention comprises a step for measuring the concentration in the blood, plasma, serum or urine of a subject suspected of renal failure of a pair of D-form and L-form of at least one amino acid selected from the amino acid group consisting of [D-serine] and [L-serine], [D-histidine] and [L-histidine], [D-asparagine] and [L-asparagine], [D-arginine] and [L-arginine], [D-allo-threonine] and [L-threonine], [D-glutamic acid] and [L-glutamic acid], [D-alanine] and [L-alanine], [D-proline] and [L-proline], [D-valine] and [L-valine], [D-allo-isoleucine] and [L-isoleucine], [D-phenylalanine] and [L-phenylalanine], and [D-lysine] and [L-lysine], and a step for calculating a pathological index value from the aforementioned concentration of the pair of D-form and L-form of aforementioned at least one amino acid.

In a different mode of the diagnostic method of the present invention, [D-glutamine] and [L-glutamine], [D-threonine] and [L-threonine], and [D-leucine] and [L-leucine] may be included in the aforementioned amino acid group. Thus, the diagnostic method of the present invention comprises a step for measuring the concentration in the blood, plasma, serum or urine of a subject suspected of renal failure of a pair of D-form and L-form of at least one amino acid selected from the amino acid group consisting of [D-serine] and [L-serine], [D-histidine] and [L-histidine], [D-asparagine] and [L-asparagine], [D-arginine] and [L-arginine], [D-allo-threonine] and [L-threonine], [D-glutamic acid] and [L-glutamic acid], [D-alanine] and [L-alanine], [D-proline] and [L-proline], [D-valine] and [L-valine], [D-allo-isoleucine] and [L-isoleucine], [D-phenylalanine] and [L-phenylalanine], [D-lysine] and [L-lysine], [D-glutamine] and [L-glutamine], [D-threonine] and [L-threonine], and [D-leucine] and [L-leucine], and a step for calculating a pathological index value from the aforementioned concentration of the pair of D-form and L-form of aforementioned at least one amino acid.

Here, the pathological index value calculated from the concentration of a pair of D-form and L-form of at least one amino acid enables a subject to be correlated with renal failure in the case of indicating a decrease in the composition ratio of the D-form. The ratio of the concentration of the D-form to the concentration of the L-form of at least one amino acid pair, or the ratio or percentage of the concentration of the D-form to the sum of the concentrations of D-form and L-form, can be used as the pathological index value of the aforementioned subject.

The diagnostic method of the present invention may further comprise a step for diagnosing that the aforementioned subject has a high likelihood of being a healthy individual or is hardly suspected of renal failure when the pathological index value of the aforementioned subject is compared with the pathological index reference value of the healthy individual and the pathological index reference value of a patient with acute renal failure and/or chronic renal failure, and the pathological index value of the aforementioned subject is similar to the pathological index reference value of the healthy individual; the aforementioned subject is diagnosed as being strongly suspected of renal failure when the pathological index value of the aforementioned subject is similar to the pathological index reference value of the aforementioned patient having acute renal failure or chronic renal failure; and the aforementioned subject is diagnosed as being suspected of early stage renal failure when the pathological index value of the aforementioned subject is between the pathological index reference value of the healthy individual and the pathological index reference value of the patient having acute renal failure or chronic renal failure.

The diagnostic method of the present invention comprises a step for diagnosing that a subject is suspected of extremely early stage renal failure when a first pathological index value of a subject calculated from the concentration in the blood, plasma, serum or urine of the aforementioned subject of a pair of D-form and L-form of at least one amino acid selected from the group consisting of [D-serine] and [L-serine], [D-histidine] and [L-histidine], [D-asparagine] and [L-asparagine], [D-arginine] and [L-arginine], [D-allo-threonine] and [L-threonine], [D-alanine] and [L-alanine], [D-proline] and [L-proline], [D-valine] and [L-valine], and [D-lysine] and [L-lysine] is between the pathological index reference value of the aforementioned healthy individual and the pathological index reference value of the aforementioned chronic renal failure patient, and a second pathological index value of the aforementioned subject calculated from the concentration in the blood, plasma, serum or urine of the aforementioned subject of a pair of D-form and L-form of at least one amino acid selected from the group consisting of [D-glutamic acid] and [L-glutamic acid], [D-allo-isoleucine] and [L-isoleucine], and [D-phenylalanine] and [L-phenylalanine] is between the pathological index reference value of the aforementioned healthy individual and the pathological index reference value of the aforementioned chronic renal failure patient.

The present invention provides a method for treating renal failure. The treatment method of the present invention comprises a step for measuring the concentration in the blood, plasma, serum or urine of a subject suspected of renal failure of a pair of D-form and L-form of at least one amino acid selected from the group consisting of [D-serine] and [L-serine], [D-histidine] and [L-histidine], [D-asparagine] and [L-asparagine], [D-arginine] and [L-arginine], [D-allo-threonine] and [L-threonine], [D-glutamic acid] and [L-glutamic acid], [D-alanine] and [L-alanine], [D-proline] and [L-proline], [D-valine] and [L-valine], [D-allo-isoleucine] and [L-isoleucine], [D-phenylalanine] and [L-phenylalanine], and [D-lysine] and [L-lysine], a step for calculating a pathological index value from the aforementioned concentration of a pair of D-form and L-form of at least one amino acid, a step for diagnosing that the aforementioned subject has a high likelihood of being a healthy individual or is hardly suspected of renal failure when the pathological index value of the aforementioned subject is compared with the pathological index reference value of the healthy individual and the pathological index reference value of a patient with acute renal failure and/or chronic renal failure and the pathological index value of the aforementioned subject is similar to the pathological index reference value of the healthy individual, the aforementioned subject is diagnosed as being strongly suspected of renal failure when the pathological index value of the aforementioned subject is similar to the pathological index reference value of the aforementioned patient having acute renal failure or chronic renal failure, and the aforementioned subject is diagnosed has being suspected of early stage renal failure when the pathological index value of the aforementioned subject is between the pathological index reference value of the healthy individual and the pathological index reference value of the patient having acute renal failure or chronic renal failure, and a step for treating the aforementioned subject when the aforementioned subject has been diagnosed as being strongly suspected of renal failure by administering a therapeutic drug that inhibits the progression or improves renal failure, including antihypertensive drugs including, but not limited to, angiotensin-converting enzymes and angiotensin II receptor antagonists, antidiabetic drugs including, but not limited to, □-glucosidase inhibitors and insulin preparations, antidyslipidemic drugs including, but not limited to, HMG-CoA reductase inhibitors and intestinal cholesterol transporter inhibitors, antianemic drugs including, but not limited to, recombinant human erythropoietin preparations, therapeutic drugs for bone and mineral metabolic disorders, therapeutic drugs for hyperuricemia, and therapeutic drugs for uremic toxins including, but not limited to, spherical adsorbent carbon medications.

The treatment method of the present invention comprises a step for diagnosing that a subject is suspected of extremely early stage renal failure when a first pathological index value of the aforementioned subject calculated from the concentration in the blood, plasma, serum or urine of the aforementioned subject of a pair of D-form and L-form of at least one amino acid selected from the group consisting of [D-serine] and [L-serine], [D-histidine] and [L-histidine], [D-asparagine] and [L-asparagine], [D-arginine] and [L-arginine], [D-allo-threonine] and [L-threonine], [D-alanine] and [L-alanine], [D-proline] and [L-proline], [D-valine] and [L-valine], and [D-lysine] and [L-lysine] is between the pathological index reference value of the aforementioned healthy individual and the pathological index reference value of the aforementioned chronic renal failure patient, and a second pathological index value of the aforementioned subject calculated from the concentration in the blood, plasma, serum or urine of the aforementioned subject of a pair of D-form and L-form of at least one amino acid selected from [D-glutamic acid] and [L-glutamic acid], [D-allo-isoleucine] and [L-isoleucine], and [D-phenylalanine] and [L-phenylalanine] is between the pathological index reference value of the aforementioned healthy individual and the pathological index reference value of the aforementioned chronic renal failure patient, and a step for treating the aforementioned subject by administering a therapeutic drug for hyperkalemia, including, but not limited to, sodium polystyrene sulfonate, and a therapeutic drug for hyperphosphatemia, including, but not limited to, calcium carbonate and calcium acetate.

Measurement of D-amino acid concentration in blood, plasma, serum or urine in the present invention may be performed using any method commonly known among persons with ordinary skill in the art. For example, a method consisting of preliminarily stereospecifically derivatizing D- and L-amino acids with o-phthaldehyde (OPA), N-tert-butyloxycarbonyl-L-cysteine (Boc-L-Cys) or other modifying reagent, followed by separating a mixture of 100 mM acetate buffer (pH 6.0) and acetonitrile by gradient elution using an analytical column in the manner of ODS-80TsQA can be used to simultaneous measure the D-forms and L-forms of aspartic acid, serine and alanine. In addition, a method consisting of preliminarily derivatizing D- and L-amino acids with a fluorescent reagent in the manner of 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and then stereospecifically separating each amino acid using an analytical column in the manner of ODS-80TsQA or Mightysil RP-18GP, followed by stereospecifically separating by optically resolving using a Pirkle chiral stationary phase column (such as the Sumichiral OA-2500S or R), can be used to measure trace amounts of proline, leucine and other amino acids (Hamase, K. and Zaitsu, K.: Analytical Chemistry, Vol. 53, 677-690 (2004)). An optical resolution column system in the present description refers to a separation analysis system that at least uses an optical resolution column, and may include separation analyses using an analytical column other than an optical resolution column. More specifically, the concentrations of D- and L-amino acids in a sample can be measured by using an optical isomer analysis method comprising a step for passing a sample containing components having optical isomers through a stationary phase in the form of a first column packing material together with a mobile phase in the form of a first liquid to separate the aforementioned components of the aforementioned sample, a step for individually retaining each of the aforementioned components of the aforementioned sample in a multi-loop unit, a step for supplying each of the aforementioned components of the aforementioned sample individually retained in the aforementioned multi-loop unit by passing the flow path through a stationary phase in the form of a second column packing material having an optically active center together with a mobile phase in the form of a second liquid to resolve the aforementioned optical isomers contained in each component of the aforementioned sample, and a step for detecting the aforementioned optical isomers contained in each component of the aforementioned sample (Japanese Patent No. 4291628). Alternatively, D-amino acids can be quantified by an immunochemical method that uses monoclonal antibodies that identify optical isomers of amino acids, such as a monoclonal antibody that specifically binds to D-leucine or D-asparagine and the like (Japanese Patent Application No. 2008-27650).

In the present description, notations of amino acids enclosed in brackets ([ ]) (such as [D-serine]) refer to the concentration of that amino acid. In the present invention, the ratio of the concentration of a D-form to the concentration of an L-form and the percentage of the concentration of a D-form to the sum of the concentrations of a D-form and an L-form, for example, are used as parameters (pathological index values) based on the concentration of an amino acid. The volume of a liquid in the manner of blood, serum, plasma or urine is reduced in order to divide the concentration of a certain substance by the concentration of another substance. Consequently, differing from the case of concentration, these parameters offer the advantage of eliminating the need for correction for liquid volume.

In the present invention, the function of the kidneys, which produce urine by specifically removing only a portion of the components in blood by filtering and reabsorbing blood, play an important role. Consequently, although amino acid concentrations in the present invention may differ greatly between blood and urine, the differences between blood, serum and plasma are not that large. This is because amino acids are not known to be specifically concentrated in blood cells or blood clots and the like. Therefore, although serum concentrations are measured in examples of the present invention and parameters based on serum concentrations are used for the pathological index values of subjects or the pathological index reference values of healthy individuals and renal failure patients, blood concentrations or plasma concentrations may be measured instead of serum concentrations, and parameters based on blood concentrations or plasma concentrations may be used for the pathological index values of subjects and the pathological index reference values of healthy individuals and renal failure patients.

In the present description, a “pathological index value” refers to a numerical value able to be calculated based on the concentrations of a plurality of biomarkers and not on the concentration of individual biomarker molecules. A pathological index value used in the present description can be calculated from the concentration of a certain D-form and L-form of at least one amino acid//, and refers to a value that enables a subject to be correlated with renal failure in the case of indicating a decrease in the composition ratio of the D-form. Pathological index values include, but are not limited to, the concentration ratio between a certain amino acid and an enantiomer thereof, such as the ratio of the concentration of a D-form to the concentration of the L-form of a certain amino acid, and the percentage of the concentration of a D-form to the sum of the concentrations of the D-form and L-form.

In the present description, a “pathological index reference value” refers to the average value or median value of the pathological index value of a biomarker molecule obtained for healthy individuals and acute renal failure and/or chronic renal failure patients diagnosed by a known diagnostic technique. Although a pathological index value is a numerical value of a specific subject at a specific point in time, a pathological index reference value is a numerical value obtained by statistical processing from a plurality of healthy individuals and patients with acute renal failure and/or chronic renal failure. Thus, a certain pathological index value being similar to a certain pathological index reference value refers to the absence of a significant difference between the pathological index value and the aforementioned pathological index reference value. For example, a statistical technique such as the two-tailed Student's t-test, one-way analysis of variance or Tukey's multiple comparison test can be used. In addition, a significance threshold P value of less than 0.05 constitutes significance in these tests.

The system for analyzing the blood, plasma, serum or urine of a subject in the present invention comprises a memory unit, analysis and measurement unit, data processing unit and pathological information output unit. Here, the memory unit contains memory that stores pathological index reference values, obtained from parameters based on enantiomer concentration data of amino acids present in blood, plasma, serum or urine, obtained for healthy individuals and patients with acute renal failure and/or chronic renal failure diagnosed by a known diagnostic technique. The aforementioned storage unit may contain significance threshold P data obtained by statistical processing from the number of the aforementioned healthy individuals and patients and individual data. The aforementioned analysis and measurement unit contains an automated analyzer, capable of automatically operating a two-dimensional HPLC system by remote control that measures amino acid enantiomer concentration explained in the present description, and a central control device for controlling the automated analyzer. The aforementioned data processing unit calculates parameters explained in the present description from amino acid enantiomer concentrations obtained with the aforementioned analysis and measurement unit. This unit also compares a pathological index value of a subject obtained from these parameters with pathological index reference values of healthy individuals and patients recalled from the aforementioned memory unit. When the pathological index value of the aforementioned subject is similar to the pathological index reference value of the aforementioned healthy individuals, information that the aforementioned subject is weakly suspected of renal failure is defined as pathological information of the aforementioned subject. When the pathological index value of the aforementioned subject is similar to the pathological index reference value of the aforementioned chronic renal failure patients, information that the aforementioned subject is suspected of renal failure is defined as pathological information of the aforementioned subject. When the pathological index value of the aforementioned subject is between the pathological index reference value of the aforementioned healthy individuals and the pathological index reference value of the aforementioned chronic renal failure patients, information that the aforementioned subject is suspected of early stage renal failure is defined as pathological information of the aforementioned subject. At this time, the aforementioned significance threshold P value may be recalled from the aforementioned memory unit and used to determine the degree to which the pathological index value of the aforementioned subject is similar to the pathological index reference value of the aforementioned healthy individuals or patients. The data processing unit contains a computer of the prior art and pathological information processing software stored in the computer. The aforementioned pathological information output unit may display the pathological information of the aforementioned subject on a liquid crystal or other display screen, output the information to a printer for printout, or transmit the pathological information of the subject via the Internet or LAN and the like as data.

In the present invention, analyzed or detected renal failure refers to a state in which renal function is depressed below that when normal, and includes all forms of kidney damage used in the normal sense of the word. Although not limited thereto, renal failure generally refers to a state in which renal function is below 30% of normal renal function, and is broadly classified into acute renal failure and chronic renal failure. Examples of the causes of depressed renal function include multiple factors such as immune system abnormalities or drug allergies, hypertension, diabetes, hemorrhage or sudden drop in blood pressure, infection or dehydration accompanying burns. Classifications of disease stage in the manner of the RIFLE classification, AKIN classification or KDIGO classification have been advocated for acute renal failure (AKI), and acute renal failure has been classified as risk (stage 1), injury (stage 2) and failure (stage 3), and as loss and end-stage kidney disease corresponding to the duration thereof. These classifications all use serum creatinine level and urine volume as indicators, and the diagnostic criterion in the case of risk (stage 1), for example, consists of a 1.5-fold to 2.0-fold increase in serum creatinine from the baseline or urine volume of less than 0.5 ml/kg/hr persisting for 6 hours or more, that in the case of injury (stage 2) consists of a 2.0-fold to 3.0-fold increase in serum creatinine from the baseline or urine volume of less than 0.5 ml/kg/hr persisting for 12 hours or more, while that in the case of failure (stage 3) consists of a 3.0-fold or more increase in serum creatinine from the baseline or urine volume of less than 0.3 ml/kg/hr persisting for 24 hours or more. On the other hand, these classifications are able to more accurately classify acute renal failure by using in combination with other indicators such as the amount of change in GFR. Diagnostic criteria for disease stage 1 (normal renal function although kidney damage is present, eGFR □90) to disease stage 5 (renal failure, eGFR<15) are indicated for chronic kidney disease (CKD) in guidelines of the Japanese Society of Nephrology (2009). Here, estimated glomerular filtration rate (eGFR), used as an indicator, is calculated with serum creatinine level based on age and gender, and indicates the capacity of the kidney to discharge body waste into urine. In the analysis and test method of the present invention, depression of renal function can be detected with higher sensitivity than conventional renal function markers. Thus, subjects can be classified into a risk group that was not classified as renal failure using conventional markers, and for example, depression of renal function can even be detected in a high risk group having risk factors for AKI or CKD in the manner of the previously described causes, but for which there are no well-defined fluctuations observed for serum creatinine or GFR.

In the present invention, the subject is not limited to a human, but rather can include experimental animals such as mice, rats, rabbits, dogs or monkeys. Thus, a subject may also be represented as a subject.

The analysis method of the present invention can be used to gather preliminary data for a method for diagnosing chronic renal failure and/or acute renal failure. Although a physician can diagnose chronic renal failure and/or acute renal failure using such preliminary data, this analysis method may also be performed by a non-physician such as a medical assistant, or can be performed by an analysis facility and the like. Thus, the analysis method of the present invention can also be said to be a preliminary diagnostic method.

All documents mentioned in the present description are incorporated in their entirety in the present description by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1-A is a typical chromatogram obtained by two-dimensional HPLC of D-/L-serine in the serum of C57BL/6J wild-type mice that underwent sham surgery or ischemia reperfusion treatment.

FIG. 1-B is a graph showing changes in D-serine concentration in the serum of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 1-C is a graph showing changes in L-serine concentration in the serum of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 1-D is a graph showing changes in the ratio of D-serine concentration to L-serine concentration in the serum of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 1-E is a graph showing changes in creatinine concentration in the serum of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 1-F is a graph showing changes in cystatin C concentration in the serum of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 2-A is a typical chromatogram obtained by two-dimensional HPLC of D-/L-serine concentration in the urine of C57BL/6J wild-type mice that underwent sham surgery and ischemia reperfusion treatment.

FIG. 2-B is a graph showing changes in D-serine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 2-C is a graph showing changes in L-serine concentration urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 2-D is a graph showing changes in creatinine concentration urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 2-E is a graph showing changes in the ratio of D-serine concentration to L-serine concentration urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 2-F is a graph showing changes in KIM-1 concentration urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 2-G is a graph showing changes in NGAL concentration urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 3-A is a graph showing changes in the ratio of D-histidine concentration to L-histidine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 3-B is a graph showing changes in the ratio of D-asparagine concentration to L-asparagine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 3-C is a graph showing changes in the ratio of D-serine concentration to L-serine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 3-D is a graph showing changes in the ratio of D-arginine concentration to L-arginine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 3-E is a graph showing changes in the ratio of D-allo-threonine concentration to L-threonine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 3-F is a graph showing changes in the ratio of D-glutamic acid concentration to L-glutamic acid concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 3-G is a graph showing changes in the ratio of D-alanine concentration to L-alanine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 3-H is a graph showing changes in the ratio of D-proline concentration to L-proline concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 3-I is a graph showing changes in the ratio of D-valine concentration to L-valine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 3-J is a graph showing changes in the ratio of D-allo-isoleucine concentration to L-isoleucine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 3-K is a graph showing changes in the ratio of D-phenylalanine concentration to L-phenylalanine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 3-L is a graph showing changes in the ratio of D-lysine concentration to L-lysine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 4-A is a graph showing changes in the percentage of D-histidine concentration to the sum of L-histidine concentration and D-histidine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 4-B is a graph showing changes in the percentage of D-asparagine concentration to the sum of L-asparagine concentration and D-asparagine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 4-C is a graph showing changes in the percentage of D-serine concentration to the sum of L-serine concentration and D-serine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 4-D is a graph showing changes in the percentage of D-arginine concentration to the sum of L-arginine concentration and D-arginine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 4-E is a graph showing changes in the percentage of D-allo-threonine concentration to the sum of L-threonine concentration and D-allo-threonine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 4-F is a graph showing changes in the percentage of D-glutamic acid concentration to the sum of L-glutamic acid concentration and D-glutamic acid concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 4-G is a graph showing changes in the percentage of D-alanine concentration to the sum of L-alanine concentration and D-alanine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 4-H is a graph showing changes in the percentage of D-proline concentration to the sum of L-proline concentration and D-proline concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 4-I is a graph showing changes in the percentage of D-valine concentration to the sum of L-valine concentration and D-valine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 4-J is a graph showing changes in the percentage of D-allo-isoleucine concentration to the sum of L-isoleucine concentration and D-allo-isoleucine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 4-K is a graph showing changes in the percentage of D-phenylalanine concentration to the sum of L-phenylalanine concentration and D-phenylalanine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 4-L is a graph showing changes in the percentage of D-lysine concentration to the sum of L-lysine concentration and D-lysine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 5-A is a graph showing changes in the ratio of D-histidine concentration to L-histidine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 5-B is a graph showing changes in the ratio of D-asparagine concentration to L-asparagine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 5-C is a graph showing changes in the ratio of D-serine concentration to L-serine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 5-D is a graph showing changes in the ratio of D-arginine concentration to L-arginine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 5-E is a graph showing changes in the ratio of D-allo-threonine concentration to L-threonine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 5-F is a graph showing changes in the ratio of D-glutamic acid concentration to L-glutamic acid concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 5-G is a graph showing changes in the ratio of D-alanine concentration to L-alanine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 5-H is a graph showing changes in the ratio of D-proline concentration to L-proline concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 5-I is a graph showing changes in the ratio of D-valine concentration to L-valine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 5-J is a graph showing changes in the ratio of D-allo-isoleucine concentration to L-isoleucine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 5-K is a graph showing changes in the ratio of D-phenylalanine concentration to L-phenylalanine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 5-L is a graph showing changes in the ratio of D-lysine concentration to L-lysine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 5-M is a graph showing changes in the ratio of D-glutamine concentration to L-glutamine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 5-N is a graph showing changes in the ratio of D-threonine concentration to L-threonine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 5-O is a graph showing changes in the ratio of D-methionine concentration to L-methionine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 5-P is a graph showing changes in the ratio of D-aspartic acid concentration to L-aspartic acid concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 5-Q is a graph showing changes in the ratio of D-allo-threonine concentration to L-allo-threonine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

FIG. 5-R is a graph showing changes in the ratio of D-leucine concentration to L-leucine concentration in the urine of C57BL/6J wild-type mice that underwent renal ischemia reperfusion treatment.

BEST MODE FOR CARRYING OUT THE INVENTION

Examples of the present invention explained below are only intended to be exemplary and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the description of the scope of claim for patent. The present invention can be modified, such as by adding, deleting or substituting constituent features of the present invention, under the condition that such modifications do not deviate from the gist of the present invention.

Example 1

1. Materials and Methods

(1) Research Ethics

All experiments were performed in accordance with facility guidelines and were approved by the animal experimentation ethics committee of the facility.

(2) Materials Amino acid enantiomers and HPLC-grade acetonitrile were purchased from Nacalai Tesque, Inc., Kyoto, Japan. HPLC-grade methanol, trifluoroacetic acid and boric acid were purchased from Wako Pure Chemical Industries, Ltd., Osaka, Japan. Water was purified using the Milli-Q Gradient A10 System.

(3) Animals

Animals were housed in an SPF environment under a light/dark cycle of 12 hours each while allowing unrestricted access to water and feed. C57BL/6J mice were purchased from CLEA Japan, Inc., Tokyo, Japan. Mice having a point mutation of D-amino acid oxidase used in the examples were the result of a mutation in which glycine at position 181 was replaced with arginine and obtained by backcrossing strain ddY mice to strain C57BL/6J mice (Sasabe, J. et al, Proc. Natl. Acad. Sci. U.S.A., 109:627 (2012)). Serine racemase knockout mice were produced according to Miyoshi, Y. et al (Amino Acids 43:1919 (2012)).

(4) Renal Ischemia Reperfusion Treatment

12- to 16-week old male mice were subjected to renal ischemia reperfusion injury (IRI). The right kidney was removed under pentobarbital anesthesia prior to IRI treatment. Mice were randomly selected after 12 days and subjected to a sham surgery or IRI treatment. The left kidney was placed outside the body and the arteries and veins were occluded with clamps (Schwartz Micro Serrefines, Fine Science Tools Inc., Vancouver, Canada). Blood circulation was resumed 45 minutes later and the clamps were removed. The return of the surface of the kidney to its original color was confirmed visually after which the kidney was returned to the body. Although the left kidney was placed outside the body in the sham surgery, occlusion of blood flow by clamping was not performed. The mice were anesthetized with diethyl ether, blood was collected from the vena cava, and urine was collected from the urinary bladder after reperfusing for 4, 8, 20 and 24 hours. Following excision, the kidneys were perfused and fixed as necessary. Serum was separated by centrifuging at 1500 □g for 10 minutes in a Becton Dickinson (BD) Microtainer. Serum and urine creatinine levels and blood urea nitrogen (BUN) levels were measured using the Fuji DRI-CHEM4000 System (Fujifilm Corp., Tokyo, Japan).

Serum cystatin C levels and urine KIM-1 and NGAL levels were measured using a mouse ELISA kit available from R&D Systems, Inc.

(5) Complete Analysis of Amino Acid Stereoisomers

The aforementioned samples were subjected to complete analysis of amino acid stereoisomers using the D/L-Amino Acid Simultaneous High-Sensitivity Analysis System developed by Zaitsu et al. Details of the analysis conditions for each amino acid are explained in Miyoshi, Y. et al, J. Chromatogr. B, 879:3194 (2011) and Sasabe, J. et al, Proc. Natl. Acad. Sci. U.S.A., 109:627 (2012). Briefly speaking, amino acids present in serum and urine were derivatized with NBD-F (4-fluoro-7-nitro-2,1,3-benzoxadiazole, Tokyo Chemical Industry Co., Ltd.) and applied to an HPLC system (refer to supplementary information provided with Nanospace SI-2, Shiseido Japan Co., Ltd.). Briefly speaking, an in-house manufactured monolithic ODS column (internal diameter: 1.5 mm □ 250 mm, installed in quartz glass capillary tube) was used for the reversed-phase separation analytical column. Fluorescence was detected at an excitation wavelength of 470 nm and detection wavelength of 530 nm. The samples were transferred to an enantiomer selective column following reversed-phase separation. The Sumichiral OA-2500S column (250 mm□ 1.5 mm, packed in-house, material manufactured by Sumika Chemical Analysis Service, Ltd.) using (S)-naphthylglycine for the chiral center was used for enantiomer separation. Concentrations of D-amino acids in body fluids were maintained on the physiological micromole order. The two-dimensional HPLC system explained in the examples is able to quantitatively measure within a range of 1 fmol to 100 pmol by distinguishing stereoisomers of serine, for example. This sensitivity was sufficient for identifying changes in the concentrations of the D-form and L-form of serine in healthy individuals and renal failure patients (not shown in the drawings).

(6) Statistical Processing

All numerical values described in the present description and drawings are indicated as the standard error of the mean±sample mean (SEM). Statistical techniques such as the two-tailed Student's t-test, one-way analysis of variance (one way ANOVA) or Tukey's multiple comparison test were used for statistical analysis of experiment results. In addition, P values of less than 0.05 were evaluated as constituting a significant difference in these tests. Prism5 (GraphPad Software, La Hoya, California) was used for all analyses.

2. Results

(1) Serum D-Serine and L-Serine Concentrations

FIG. 1-A is a typical chromatogram obtained by two-dimensional HPLC of D-/L-serine in the serum of C57BL/6J wild-type mice that underwent sham surgery or ischemia reperfusion treatment. In the following experiment, markers were measured for 8 animals in a sham group and for 5, 9, 6 and 7 animals at 4, 8, 20 and 40 hours, respectively, after reperfusion. The bar graphs of FIGS. 1-A to 1-F represent average values, while the error bars represent the standard error of the sample mean (SEM). Data of the examples was tested statistically by one way analysis of variance followed by Tukey's multiple comparison test. In FIGS. 1-A to 1-F, one asterisk (*) indicates a P value of less than 0.05, two asterisks (**) indicate a P value of less than 0.01, and three asterisks (***) indicate a P value of less than 0.001. NS stands for not significant. The word “sham” in the drawings indicates concentrations in mice that underwent sham surgery, while IRI4, IRI8, IRI20 and IRI40 indicate concentrations in mice at 4, 8, 20 and 40 hours after reperfusion, respectively. Although there were significant fluctuations in serum D-serine concentrations at 4 and 8 hours after reperfusion in the C57BL/6J mice, concentrations increased at 20 hours and increased further at 40 hours (FIG. 1-B). Furthermore, the values of D-serine concentration indicated in FIG. 1-B were 3.7±0.4 μM in the sham surgery mice, 3.4±0.3 μM for IRI4, 4.3±0.4 μM for IRI8, 5.5±0.5 μM for IRI20 and 10.6±0.4 μM for IRI40. Serum L-serine concentrations decreased 4 hours after reperfusion and subsequently remained at a low value (FIG. 1-C). The values of L-serine concentration indicated in FIG. 1-C were 106.1±5.6 μM in the sham surgery mice, 46.9±0.6 μM for IRI4, 61.5±5.6 μM for IRI8, 70.6±7.5 μM for IRI20 and 64.7±2.2 μM for IRI40. Consequently, the ratio of [D-serine]/[L-serine] increased accompanying the decrease in L-serine concentration and increased further after 40 hours (FIG. 1-D). The values of [D-serine]/[L-serine] shown in FIG. 1-D were 0.036±0.004 in the sham surgery mice, 0.074±0.005 for IRI4, 0.073±0.009 for IRI8, 0.082±0.009 for IRI20 and 0.164±0.008 for IRI40. Serum creatinine concentrations increased starting 4 hours after reperfusion and increased further 40 hours after reperfusion (FIG. 1-E). The values of creatinine concentration indicated in FIG. 1-E were 0.59±0.05 mg/dl in the sham surgery mice, 1.108±0.04 mg/dl for IRI4, 1.89±0.09 mg/dl for IRI8, 1.14±0.22 mg/dl for IRI20 and 3.73±0.09 mg/dl for IRI40. However, serum cystatin C concentrations gradually decreased after 40 hours after having initially increased at 4 hours after reperfusion (FIG. 1-F). The values of cystatin C concentration indicated in FIG. 1-F were 0.84±0.01 μg/ml in the sham surgery mice, 1.63±0.08 μg/ml for IRI4, 1.39±0.09 μg/ml for IRI8, 1.19±0.05 μg/ml for IRI20 and 1.06±0.10 μg/ml for IRI40. On the basis of these experiments, the ratio of [D-serine]/[L-serine] began to increase 4 hours after reperfusion and then exhibited a monotonic increase until 40 hours after reperfusion, thereby clearly indicating that it is useful as a marker of renal failure. Here, although a certain value is only indicated at a certain point in time after reperfusion in the case of a monotonically changing marker, in the case of fluctuations having a peak and trough, a certain value is not only indicated at a single point in time, but may also increase another time or a plurality of times more. Consequently, the stage of progression of renal failure cannot be uniquely estimated by the value of a marker.

(2) Urine D-Serine and L-Serine

In the following experiment, markers were measured for 7 animals in a sham group and for 5 animals each at 4, 8, 20 and 40 hours after reperfusion. The bar graphs of FIGS. 2-A to 2-J represent average values, while the error bars represent the standard error of the sample mean (SEM). Data of the examples was tested statistically by one way analysis of variance followed by Tukey's multiple comparison test. In FIGS. 2-A to 2-G, one asterisk (*) indicates a P value of less than 0.05, two asterisks (**) indicate a P value of less than 0.01, and three asterisks (***) indicate a P value of less than 0.001. NS stands for not significant. Although serum D-serine concentrations increased with the passage of time after reperfusion, L-serine concentrations decreased. In urine, however, D-serine concentrations conversely decreased with the passage of time after reperfusion (FIG. 2-B), while L-serine concentrations increased (FIG. 2-C). The values of D-serine concentration indicated in FIG. 2-B were 52.0±7.6 μM in the sham surgery mice, 24.5±5.7 μM for IRI4, 9.9±1.1 μM for IRI8, 36.9±3.3 μM for IRI20 and 22.4±3.8 μM for IRI40. The values of L-serine indicated in FIG. 2-C were 19.0±3.0 μM in the sham surgery mice, 23.6±2.7 μM for IRI4, 62.6±9.9 μM for IRI8, 136.1±14.9 μM for IRI20 and 93.8±12.1 μM for IRI40. Furthermore, urine creatinine levels decreased starting 8 hours after reperfusion (FIG. 2-D). This is due to outflow of creatinine into the urine having been inhibited by depressed renal function. Although depression of renal function was not remarkable until 4 hours after reperfusion since creatinine levels did not differ that much from the sham surgery mice at 4 hours after reperfusion, the ratio of [D-serine]/[L-serine] in urine decreased to nearly one-third of that of the sham surgery mice after 4 hours (FIG. 2-E). Therefore, the ratio of [D-serine]/[L-serine] in urine fluctuated prior to depression of renal function, and since it decreased monotonically, it was indicated to be useful as an early marker of renal failure. The values of the ratio of [D-serine]/[L-serine] indicated in FIG. 2-E were 2.82±0.18 in the sham surgery mice, 1.10±0.26 for IRI4, 0.16±0.01 for IRI8, 0.28±0.02 for IRI20 and 0.25±0.04 for IRI40. Although urine KIM-1 concentrations increased through 20 hours after reperfusion, they decreased at 40 hours after reperfusion (FIG. 2-F). Urine NGAL concentrations did not differ significantly from the sham surgery mice at 4 hours after reperfusion, increased at 8 hours and subsequently remained substantially unchanged (FIG. 2-G). Thus, parameters based on urine serine concentration demonstrated fluctuations accompanying renal failure that started earlier than any of the known markers, and since those fluctuations changed monotonically, they are useful for determining the stage of the progression of renal failure of a subject.

(3) Changes in Concentration Ratios of Various Amino Acid Enantiomers in Urine

The concentrations of various amino acid enantiomer pairs were measured for the urine of two of the mice used in the experiment of FIG. 2-A to 2-J in which urine markers were measured. FIGS. 3-A to 3-L indicate bar graphs of the ratio of the average value of the concentration of a D-form to the average value of the concentration of an L-form of individual mice for the mice at 4, 8, 20 and 40 hours after ischemia reperfusion treatment. As a result, in the case of [D-glutamic acid] and [L-glutamic acid] (FIG. 3-F), [D-allo-isoleucine] and [L-isoleucine] (FIG. 3-J) and [D-phenylalanine] and [L-phenylalanine](FIG. 3-K) in urine, the ratio of the concentration of the D-form to the concentration of the L-form did not fluctuate 4 hours after reperfusion, decreased considerably starting 8 hours after reperfusion. In contrast, in the combinations of [D-histidine] and [L-histidine] (FIG. 3-A), [D-asparagine] and [L-asparagine] (FIG. 3-B), [D-serine] and [L-serine] (FIG. 3-C), [D-arginine] and [L-arginine] (FIG. 3-D), [D-allo-threonine] and [L-threonine] (FIG. 3-E), [D-alanine] and [L-alanine] (FIG. 3-G), [D-proline] and [L-proline] (FIG. 3-H), [D-valine] and [L-valine] (FIG. 3-I) and [D-lysine] and [L-lysine] (FIG. 3-L), the ratio of the concentration of the D-form to the concentration of the L-form fluctuated considerably at 4 hours after reperfusion, demonstrating values intermediate to the sham surgery mice and values starting 8 hours after reperfusion. Therefore, if at least one of any of [D-histidine]/[L-histidine], [D-asparagine]/[L-asparagine], [D-arginine]/[L-arginine], [D-allo-threonine]/[L-threonine], [D-alanine]/[L-alanine], [D-proline]/[L-proline], [D-valine]/[L-valine] and [D-lysine]/[L-lysine] in a certain individual is lower than the value of a healthy individual, even if at least any one of [D-glutamic acid]/[L-glutamic acid], [D-allo-i-allo-isoleucine]/[L-isoleucine] and [D-phenylalanine]/[L-phenylalanine] has a value that is no different from the value of a healthy individual, an extremely early state prior to the onset of depression of renal function can be detected. In addition, when at least one of any of [D-histidine]/[L-histidine], [D-asparagine]/[L-asparagine], [D-arginine]/[L-arginine], [D-allo-threonine]/[L-threonine], [D-alanine]/[L-alanine], [D-proline]/[L-proline], [D-valine]/[L-valine] and [D-lysine]/[L-lysine] in a certain individual is lower than the value of a healthy individual, and at least any one of [D-glutamic acid]/[L-glutamic acid], [D-allo-isoleucine]/[L-isoleucine] and [D-phenylalanine]/[L-phenylalanine] is lower than the value of a healthy individual, a state at a time when depression of renal function has begun is detected. In this manner, not only whether or not a subject is in the early stage of renal failure, but also an extremely early stage prior to the onset of depression of renal function, or even a state at the time depression of renal function has begun, can be distinguished by parameters based on the urine concentrations of the D-forms and L-forms of different groups of amino acids.

(4) Changes in Percentage of Concentration of D-Form to the Total

Concentrations of Various Amino Acid Enantiomers in Urine FIGS. 4-A to 4-L indicate bar graphs of the percentages of the average values of concentrations of a D-form to the sum of the average values of the concentrations of an L-form and the average values of the concentrations of the D-form of individual mice for sham surgery mice and mice 4, 8, 20 and 40 hours after ischemia reperfusion treatment. As a result, in the case of [D-glutamic acid] and [L-glutamic acid] (FIG. 4-F), [D-allo-isoleucine] and [L-isoleucine] (FIG. 4-J) and [D-phenylalanine] and [L-phenylalanine] (FIG. 4-K) in urine, the percentage of the average value of the concentration of the D-form to the sum of the average value of the concentration of the L-form and the average value of the concentration of D-form did not fluctuate even at 4 hours after reperfusion, and decreased considerably starting 8 hours after reperfusion. In contrast, in the combinations of [D-histidine] and [L-histidine] (FIG. 4-A), [D-asparagine] and [L-asparagine] (FIG. 4-B), [D-serine] and [L-serine] (FIG. 4-C), [D-arginine] and [L-arginine] (FIG. 4-D), [D-allo-threonine] and [L-threonine] (FIG. 4-E), [D-alanine] and [L-alanine] (FIG. 4-G), [D-proline] and [L-proline] (FIG. 4-H), [D-valine] and [L-valine] (FIG. 4-I) and [D-lysine] and [L-lysine] (FIG. 4-L), the percentage of the average value of the concentration of the D-form to the sum of the average value of the concentration of the L-form and the average value of the concentration of the D-form fluctuated considerably 4 hours after reperfusion, demonstrating values intermediate to the sham surgery mice and values starting 8 hours after reperfusion. Therefore, if at least one of any of the percentage of [D-histidine] to [total histidine], the percentage of [D-asparagine] to [total asparagine], the percentage of [D-arginine] to [total arginine], the percentage of [D-allo-threonine] to the sum of [D-allo-threonine] and [L-threonine], the percentage of [D-alanine] to [total alanine], the percentage of [D-proline] to [total proline], the percentage of [D-valine] to [total valine], and the percentage of [D-lysine] to [total lysine] in a certain individual is lower than the value of a healthy individual, even if at least any one of the percentage of [D-glutamic acid] to [total glutamic acid], the percentage of [D-allo-isoleucine] to the sum of [D-allo-isoleucine] and [L-isoleucine] and the percentage of [D-phenylalanine] to [total phenylalanine] is no different from the value of a healthy individual, an extremely early state prior to the onset of depression of renal function can be detected.

In addition, when at least one of any of the percentage of [D-histidine] to [total histidine], the percentage of [D-asparagine] to [total asparagine], the percentage of [D-arginine] to [total arginine], the percentage of [D-allo-threonine] to the sum of [D-allo-threonine] and [L-threonine], the percentage of [D-alanine] to [total alanine], the percentage of [D-proline] to [total proline], the percentage of [D-valine] to [total valine], and the percentage of [D-lysine] to [total lysine] in a certain individual is lower than the value of a healthy individual, and at least one of any of the percentages of [D-glutamic acid] to [total glutamic acid], the percentage of [D-allo-isoleucine] to the sum of [D-allo-isoleucine] and [L-isoleucine] and the percentage of [D-phenylalanine] to [total phenylalanine] is also lower than the value of a healthy individual, a state at a time when depression of renal function has begun is detected. In this manner, not only whether or not a subject is in the early stage of renal failure, but also an extremely early stage prior to the onset of depression of renal function, or even a state at the time depression of renal function has begun, can be distinguished by parameters based on the urine concentrations of the D-forms and L-forms of different groups of amino acids.

-   -   (5) Changes in Concentration Ratios of Various Amino Acid         Enantiomers in Urine

Ischemia reperfusion treatment was performed on 3 to 7 mice and the concentrations of various amino acid enantiomer pairs were measured in the acquired urine. FIGS. 5-A to 5-R indicate bar graphs of the ratios of the average value of the concentration of a D-form to the average value of the concentration of an L-form in individual mice in sham surgery mice and mice at 4, 8, 20 and 40 hours after ischemia reperfusion treatment along with the results of investigating for the presence or absence of statistically significant differences. As a result, in the case of [D-allo-isoleucine] and [L-isoleucine] (FIG. 5-J), [D-phenylalanine] and [L-phenylalanine] (FIG. 5-K) and [D-leucine] and [L-leucine] (FIG. 5-R) in urine, the ratio of the concentration of the D-form to the concentration of the L-form did not fluctuate even at 4 hours after reperfusion (absence of significant difference) and decreased considerably starting 8 hours after reperfusion (presence of significant difference). In the case of [D-glutamic acid] and [L-glutamic acid] (FIG. 5-F), [D-valine] and [L-valine] (FIG. 5-I), [D-glutamine] and [L-glutamine] (FIG. 5-M), [D-threonine] and [L-threonine] (FIG. 5-N) and [D-allo-threonine] and [L-allo-threonine] (FIG. 5-Q), although values fluctuated even at 4 hours after reperfusion, there were no statistically significant differences, while values decreased considerably starting at 8 hours after reperfusion (presence of significant difference). In the case of [D-methionine] and [L-methionine] (FIG. 5-O) and [D-aspartic acid] and [L-aspartic acid] (FIG. 5-P), there were no fluctuating tendencies observed. In contrast, in the combinations of [D-histidine] and [L-histidine] (FIG. 5-A), [D-asparagine] and [L-asparagine] (FIG. 5-B), [D-serine] and [L-serine] (FIG. 5-C), [D-arginine] and [L-arginine](FIG. 5-D), [D-allo-threonine] and [L-threonine] (FIG. 5-E), [D-alanine] and [L-alanine] (FIG. 5-G), [D-proline] and [L-proline] (FIG. 5-H) and [D-lysine] and [L-lysine] (FIG. 5-L), the ratio of the concentration of the D-form to the concentration of the L-form fluctuated considerably at 4 hours after perfusion (presence of statistically significant difference), demonstrating values intermediate to the sham surgery mice and values starting at 8 hours after reperfusion. In the case of urine creatinine conventionally used as a diagnostic marker of renal failure, since renal failure was unable to be detected at 4 hours after reperfusion and was only able to be detected starting at 8 hours after reperfusion in a ischemia reperfusion model using mice (FIG. 2-D), any of the [D-allo-isoleucine] and [L-isoleucine], [D-phenylalanine] and [L-phenylalanine], [D-leucine] and [L-leucine], [D-glutamic acid] and [L-glutamic acid], [D-valine] and [L-valine], [D-glutamine] and [L-glutamine], [D-threonine] and [L-threonine], [D-allo-threonine] and [L-allo-threonine], [D-histidine] and [L-histidine], [D-asparagine] and [L-asparagine], [D-serine] and [L-serine], [D-arginine] and [L-arginine], [D-allo-threonine] and [L-threonine], [D-alanine] and [L-alanine], [D-proline] and [L-proline] (FIG. 5-H) and [D-lysine] and [L-lysine] (FIG. 5-L) can be used as a marker for renal failure having sensitivity that is equal to or greater than that of urine creatinine. If one or a plurality of pathological index values selected from the group consisting of [D-histidine]/[L-histidine], [D-asparagine]/[L-asparagine], [D-arginine]/[L-arginine], [D-allo-threonine]/[L-threonine], [D-alanine]/[L-alanine], [D-proline]/[L-proline] and [D-lysine]/[L-lysine], which are capable of detection at 4 hours after reperfusion with a significant difference in particular, is used, renal failure can be diagnosed with higher sensitivity than urine creatinine. Among these, one or a plurality of pathology index values selected from the group consisting of [D-histidine]/[L-histidine], [D-asparagine]/[L-asparagine], [D-proline]/[L-proline] and [D-lysine]/[L-lysine], which demonstrate a significant difference of P<0.01 with the sham surgery group at 4 hours after reperfusion, in particular is capable of diagnosing renal failure with higher sensitivity, while one or a plurality of pathological index values selected from the group consisting of [D-histidine]/[L-histidine], [D-proline]/[L-proline] and [D-lysine]/[L-lysine], which demonstrate a significant difference of P<0.001 with the sham surgery group at 4 hours after reperfusion, is capable of diagnosing renal failure with even higher sensitivity. Although pathological index values may be used alone, combining a plurality of pathological index values enables diagnoses having a higher level of reliability.

In addition, among the pathological index values of the present invention, pathological index values calculated using the concentrations of any of the pairs of D-forms and L-forms among [D-allo-isoleucine] and [L-isoleucine], [D-phenylalanine] and [L-phenylalanine], [D-leucine] and [L-leucine], [D-glutamic acid] and [L-glutamic acid], [D-valine] and [L-valine], [D-glutamine] and [L-glutamine], [D-threonine] and [L-threonine], [D-allo-threonine] and [L-allo-threonine], [D-histidine] and [L-histidine], [D-asparagine] and [L-asparagine], [D-serine] and [L-serine], [D-arginine] and [L-arginine], [D-allo-threonine] and [L-threonine], [D-alanine] and [L-alanine], [D-proline] and [L-proline] (FIG. 5-H) and [D-lysine] and [L-lysine] can be used as markers of renal failure having sensitivity that is equal to or greater than that of urine creatinine. Thus, in the case a pathological index value of a subject is statistically significantly different from the pathological index reference value of a healthy individual group and statistically significantly different from the pathological index reference value of a renal failure patient group, and is between the pathological index reference value of a healthy individual group and the pathological index reference value of a renal failure patient group, the subject can be diagnosed as being suspected of early renal failure. In particular, the use of a pathological index value calculated using the concentration of one or a plurality of a pair of D-form and L-form of one or more amino acids selected from the group consisting of [D-histidine] and [L-histidine], [D-asparagine] and [L-asparagine], [D-proline] and [L-proline] and [D-lysine] and [L-lysine], which exhibit a significant difference of p<0.01 with a sham surgery group at 4 hours after reperfusion, enables diagnosis of renal failure at an earlier stage. Moreover, renal failure can be diagnosed at an even earlier stage by using a pathological index value calculated from the concentration of one or a plurality of a pair of D-form and L-form of one or more amino acids selected from the group consisting of [D-histidine] and [L-histidine], [D-proline] and [L-proline] and [D-lysine] and [L-lysine], which exhibit a significant difference of p<0.001 with a sham surgery group at 4 hours after reperfusion.

ADDITIONAL DISCLOSURE Technical Field

The present invention relates to an apparatus, a system and a method for analyzing a disease sample, on the basis of a quantitative determination method that discriminates stereoisomers of an amino acid. Specifically, the present invention relates to an apparatus for analyzing a disease sample, the apparatus including: a member for separating and quantitatively determining amino acid stereoisomers in a biological material from a subject; a member for obtaining a disease state index value through a calculation by substituting a quantitatively determined value of the amino acid stereoisomer into a discriminant equation; and a member for outputting disease state information on the subject on the basis of the disease state index value. Further, the present invention also relates to a system for analyzing a disease sample, the system including: a quantitative determination analysis unit for separating and quantitatively determining an amino acid stereoisomer in a biological material from a subject; a disease state index value computation unit for obtaining a disease state index value through a calculation by substituting a quantitatively determined value of the amino acid stereoisomer into a discriminant equation; and a disease state information output unit for outputting disease state information on the subject on the basis of the disease state index value. Additionally, the present invention further relates to a method for analyzing a disease sample, the method including: a step of separating and quantitatively determining an amino acid stereoisomer in a biological material from a subject; a step of obtaining a disease state index value through a calculation by substituting a quantitatively determined value of the amino acid stereoisomer into a discriminant equation; and a step of obtaining disease state information on the subject on the basis of the disease state index value.

Background Art

There are two types of stereoisomers, i.e., D-form and L-form, of all amino acids other than glycine. L-amino acids are constitutive elements of proteins in organisms, and thus amino acids contained in proteins are L-amino acids in principle. In contrast, D-amino acids are contained in a part of bioactive peptides in lower organisms, and many of these are biosynthesized via a posttranslational modification process. Accordingly, amino acids that constitute proteins or peptides are predominantly L-amino acids, whereas D-amino acids are exceptionally present.

D-amino acids are one of constitutive components of peptide glycan of bacterial cell walls. In addition, with regard to free D-amino acids that do not constitute peptides, the presence thereof in lower animals such as aquatic animals and insects has been conventionally reported. However, it was believed in some past times that amino acids are present in their L-form in higher animals, without an involvement of D-forms in the physiological activities (Nonpatent Document 10).

However, the presence and roles of D-amino acids in mammals including humans have been managed to be elucidated in accordance with improvements of a resolution ability and sensitivity owing to the advance of analytical techniques in recent years (Nonpatent Document 11). D-aspartic acid was proven to be localized in prolactin-producing cells in rat pituitary gland by means of a double staining process and the like carried out using an anti-D-aspartic acid antibody. Also, addition of D-aspartic acid to cells that synthesize and secrete prolactin, a cell strain derived from rat pituitary gland, resulted in an increase of the secretion of prolactin in a dose-dependent manner. From these findings, it is believed that D-aspartic acid controls the secretion of prolactin in prolactin-producing cells (Nonpatent Document 12).

On the other hand, it was reported that D-aspartic acid is detected in rat testicular vein at a constantly higher concentration than that in other venous blood, and further that addition of D-aspartic acid to Leydig cells isolated and purified from rat testis promotes the synthesis and secretion of testosterone in a dose-dependent manner (Nonpatent Document 13).

It is reported that D-serine selectively stimulates a glycine binding site of an NMDA type glutamic acid receptor which is presumed to involve in schizophrenia, and promotes neurotransmission through enhancing an action of glutamic acid that is operated via this receptor (Nonpatent Document 14). In fact, it was reported that administration with D-serine ameliorates schizophrenia, and that the concentration of D-serine in sera from schizophrenia patients is lower than that from healthy individuals. Moreover, it was also reported recently that D-serine involves in degeneration of motor nerve in amyotrophic lateral sclerosis (ALS) (Nonpatent Document 15).

Zaitsu et al., developed a simultaneous and highly sensitive analysis system of D- and L-amino acids (Patent Document 2, Nonpatent Documents 16 to 18) to put a simultaneous and highly sensitive analytical technique of amino acid stereoisomers into a practical application. Accordingly, it has become possible to make comprehensive separation and quantitative determination of D-amino acids present in a trace amount and L-amino acids present in a comparatively large amount both contained in a human biological material from an identical sample.

In connection with the present invention, it was found from results of quantitative determination analyses on amino acid stereoisomers from various types of disease patients, that the amounts of D-amino acids and L-amino acids in biological materials from healthy individuals are maintained at a constant balance, with small individual differences, and that there is a disturbance in balance, in some amino acids, that cannot be ascertained unless D-amino acids and L-amino acids in biological materials from patients are separated and quantitatively determined. The present invention was accomplished in view of such unexpected findings.

PRIOR ART DOCUMENTS Patent Documents

-   Patent Document 2: Japanese Patent No. 4291628

Nonpatent Documents

-   Nonpatent Document 10: Corrigan J. J., Science 164: 142 (1969) -   Nonpatent Document 11: Hamase K, Morikawa A, and Zaitsu K., J     Chromatogr. B 781: 73 (2002) -   Nonpatent Document 12: D'Aniello A et el., FASEB J 14: 699 (2000) -   Nonpatent Document 13: Nagata Y et el., FEBS Lett. 444: 160 (1999) -   Nonpatent Document 14: Nishikawa T, Biol. Pharm. Bull. 28: 1561     (2005) -   Nonpatent Document 15: Sasabe, J., et el., Proc. Natl. Acad. Sci.     109: 627 (2012) -   Nonpatent Document 16: Hamase K., et el., J. Chromatogr. A, 1143:     105 (2007) -   Nonpatent Document 17: Hamase K., et el., J. Chromatogr. A, 1217:     1056 (2010) -   Nonpatent Document 18: Miyoshi Y., et el., J. Chromatogr. B, 879:     3184 (2011)

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

There is a need for a development of a novel method for diagnosing a disease by a total analysis of amino acid stereoisomers to elucidate a correlation between a disease and the amount of an amino acid stereoisomer or the change in the amount thereof, as well as a novel apparatus for diagnosing a disease in which the method for diagnosing a disease is executed.

Means for Solving the Problems

According to an aspect of the present invention, an apparatus for analyzing a disease sample is provided. The apparatus for analyzing a disease sample according to the aspect of the present invention includes a member for separating and quantitatively determining an amino acid stereoisomer in a biological material from a subject, a member for obtaining a disease state index value through a calculation by substituting the amount of the amino acid stereoisomer into a discriminant equation, and a member for outputting disease state information on the subject on the basis of the disease state index value.

In the apparatus for analyzing a disease sample according to the aspect of the present invention, the discriminant equation may be either:

disease state index value=(a measurement value for an amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)/(a reference value for the amino acid stereoisomer that correlates with the disease in a biological material from a healthy individual); or

disease state index value=[(a measurement value for an amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)/{(a measurement value for an amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)+(a measurement value for an enantiomer of the amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)}]÷[(a reference value for the amino acid stereoisomer that correlates with the disease from among reference values from a healthy individual)/{(a reference value for the amino acid stereoisomer that correlates with the disease from among reference values from the healthy individual)+(a reference value for the enantiomer of the amino acid stereoisomer that correlates with the disease from among reference values from the healthy individual)}].

In the apparatus for analyzing a disease sample according to the aspect of the present invention, the member for outputting disease state information on the subject on the basis of the disease state index value may be a member for outputting disease state information on the subject that the subject is suffering from the disease when the disease state index value is 2.0 or greater.

In the apparatus for analyzing a disease sample according to the aspect of the present invention, the amino acid stereoisomer that correlates with the disease may be: one, or two or more types of amino acids selected from the group consisting of D-serine, D-threonine, D-alanine, D-asparagine, allo-D-threonine, D-glutamine, D-proline and D-phenylalanine when the disease is a renal disease; D-histidine and/or D-asparagine when the disease is prostate gland cancer; D-asparagine when the disease is osteoporosis; D-serine, L-arginine, D-glutamic acid and D-proline when the disease is dilated cardiomyopathy; L-histidine, L-phenylalanine and D-aspartic acid when the disease is a climacteric disorder; D-arginine when the disease is sarcoma; D-allo-isoleucine, D-serine, D-alanine, D-methionine, D-leucine, D-aspartic acid, D-phenylalanine and L-phenylalanine when the disease is Alzheimer's disease; D-serine, D-allo-threonine, D-alanine, D-proline, D-leucine and D-phenylalanine in the case of DAO deficiency; D-asparagine, D-aspartic acid and D-arginine in the case of DDO deficiency; L-phenylalanine in the case of phenylketonuria; L-valine, L-allo-isoleucine, D-isoleucine, L-isoleucine and L-leucine in the case of a maple syrup urine disease; L-glutamic acid, L-glutamine and L-cysteine in the case of articular rheumatism; D-serine and D-alanine in the case of kidney cancer; D-alanine in the case of lung cancer; L-arginine and L-glutamic acid in the case of a cardiovascular disease; D-serine and L-cysteine in the case of multiple sclerosis; L-cysteine in the case of acute myeloid leukemia; L-cysteine in the case of lymphoma; L-glutamic acid and L-cysteine in the case of acute lymphocytic leukemia; L-arginine and L-cysteine in the case of psoriasis; or D-alanine, L-cysteine and L-glutamic acid in the case of diabetes.

According to another aspect of the present invention, a system for analyzing a disease sample is provided. The system for analyzing a disease sample according to the another aspect of the present invention includes: a quantitative determination analysis unit for separating and quantitatively determining an amino acid stereoisomer in a biological material from a subject; a disease state index value computation unit for obtaining a disease state index value through a calculation by substituting the amount of the amino acid stereoisomer into a discriminant equation; and a disease state information output unit for outputting disease state information on the subject on the basis of the disease state index value.

In the system for analyzing a disease sample according to the another aspect of the present invention, the discriminant equation may be either:

disease state index value=(a measurement value for an amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)/(a reference value for the amino acid stereoisomer that correlates with the disease in a biological material from a healthy individual); or

disease state index value=[(a measurement value for an amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)/{(a measurement value for an amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)+(a measurement value for an enantiomer of the amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)}]÷[(a reference value for the amino acid stereoisomer that correlates with the disease from among reference values from a healthy individual)/{(a reference value for the amino acid stereoisomer that correlates with the disease from among reference values from the healthy individual)+(a reference value for the enantiomer of the amino acid stereoisomer that correlates with the disease from among reference values from the healthy individual)}].

In the system for analyzing a disease sample according to the another aspect of the present invention, the disease state information output unit may output disease state information on the subject that the subject is suffering from the disease when the disease state index value is 2.0 or greater.

In the system for analyzing a disease sample according to the another aspect of the present invention, the amino acid stereoisomer that correlates with the disease may be: one, or two or more types of amino acids selected from the group consisting of D-serine, D-threonine, D-alanine, D-asparagine, allo-D-threonine, D-glutamine, D-proline and D-phenylalanine when the disease is a renal disease; D-histidine and/or D-asparagine when the disease is prostate gland cancer; D-asparagine when the disease is osteoporosis; D-serine, L-arginine, D-glutamic acid and D-proline when the disease is dilated cardiomyopathy; L-histidine, L-phenylalanine and D-aspartic acid when the disease is a climacteric disorder; D-arginine when the disease is sarcoma; D-allo-isoleucine, D-serine, D-alanine, D-methionine, D-leucine, D-aspartic acid, D-phenylalanine and L-phenylalanine when the disease is Alzheimer's disease; D-serine, D-allo-threonine, D-alanine, D-proline, D-leucine and D-phenylalanine in the case of DAO deficiency; D-asparagine, D-aspartic acid and D-arginine in the case of DDO deficiency; L-phenylalanine in the case of phenylketonuria; L-valine, L-allo-isoleucine, D-isoleucine, L-isoleucine and L-leucine in the case of a maple syrup urine disease; L-glutamic acid, L-glutamine and L-cysteine in the case of articular rheumatism; D-serine and D-alanine in the case of kidney cancer; D-alanine in the case of lung cancer; L-arginine and L-glutamic acid in the case of a cardiovascular disease; D-serine and L-cysteine in the case of multiple sclerosis; L-cysteine in the case of acute myeloid leukemia; L-cysteine in the case of lymphoma; L-glutamic acid and L-cysteine in the case of acute lymphocytic leukemia; L-arginine and L-cysteine in the case of psoriasis; or D-alanine, L-cysteine and L-glutamic acid in the case of diabetes.

According to still another aspect of the present invention, a method for analyzing a disease sample is provided. The method for analyzing a disease sample according to the still another aspect of the present invention includes: a step of measuring the amount of an amino acid stereoisomer in a biological material from a subject; a step of obtaining a disease state index value through a calculation by substituting the amount of the amino acid stereoisomer into a discriminant equation; and a step of outputting disease state information on the subject on the basis of the disease state index value.

In the method for analyzing a disease sample according to the still another aspect of the present invention, the discriminant equation may be either:

disease state index value=(a measurement value for an amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)/(a reference value for the amino acid stereoisomer that correlates with the disease in a biological material from a healthy individual); or

disease state index value=[(a measurement value for an amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)/{(a measurement value for an amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)+(a measurement value for an enantiomer of the amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)}]÷[(a reference value for the amino acid stereoisomer that correlates with the disease from among reference values from a healthy individual)/{(a reference value for the amino acid stereoisomer that correlates with the disease from among reference values from the healthy individual)+(a reference value for the enantiomer of the amino acid stereoisomer that correlates with the disease from among reference values from the healthy individual)}].

In the method for analyzing a disease sample according to the still another aspect of the present invention, the step of outputting disease state information on the subject on the basis of the disease state index value may be a step of outputting disease state information on the subject that the subject is suffering from the disease when the disease state index value is 2.0 or greater.

In the method for analyzing a disease sample according to the still another aspect of the present invention, the amino acid stereoisomer that correlates with the disease may be: one, or two or more types of amino acids selected from the group consisting of D-serine, D-threonine, D-alanine, D-asparagine, allo-D-threonine, D-glutamine, D-proline and D-phenylalanine when the disease is a renal disease; D-histidine and/or D-asparagine when the disease is prostate gland cancer; D-asparagine when the disease is osteoporosis; D-serine, L-arginine, D-glutamic acid and D-proline when the disease is dilated cardiomyopathy; L-histidine, L-phenylalanine and D-aspartic acid when the disease is a climacteric disorder; D-arginine when the disease is sarcoma; D-allo-isoleucine, D-serine, D-alanine, D-methionine, D-leucine, D-aspartic acid, D-phenylalanine and L-phenylalanine when the disease is Alzheimer's disease; D-serine, D-allo-threonine, D-alanine, D-proline, D-leucine and D-phenylalanine in the case of DAO deficiency; D-asparagine, D-aspartic acid and D-arginine in the case of DDO deficiency; L-phenylalanine in the case of phenylketonuria; L-valine, L-allo-isoleucine, D-isoleucine, L-isoleucine and L-leucine in the case of a maple syrup urine disease; L-glutamic acid, L-glutamine and L-cysteine in the case of articular rheumatism; D-serine and D-alanine in the case of kidney cancer; D-alanine in the case of lung cancer; L-arginine and L-glutamic acid in the case of a cardiovascular disease; D-serine and L-cysteine in the case of multiple sclerosis; L-cysteine in the case of acute myeloid leukemia; L-cysteine in the case of lymphoma; L-glutamic acid and L-cysteine in the case of acute lymphocytic leukemia; L-arginine and L-cysteine in the case of psoriasis; or D-alanine, L-cysteine and L-glutamic acid in the case of diabetes.

According to yet another aspect of the present invention, a method for diagnosing a disease is provided. The method for diagnosing a disease according to the yet another aspect of the present invention includes: a step of measuring the amount of an amino acid stereoisomer in a biological material from a subject; and a step of diagnosing the disease on the basis of a measurement value of the amount of the amino acid stereoisomer and a reference value from a healthy individual.

According to yet another aspect of the present invention, a method for diagnosing a disease is provided. The method for diagnosing a disease according to the yet another aspect of the present invention includes: a step of separating and quantitatively determining an amino acid stereoisomer in a biological material from a subject; a step of obtaining a disease state index value through a calculation by substituting the amount of the amino acid stereoisomer into a discriminant equation; and a step of diagnosing the subject on the basis of the disease state index value.

In the method for diagnosing a disease according to the yet another aspect of the present invention, the discriminant equation may be either:

disease state index value=(a measurement value for an amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)/(a reference value for the amino acid stereoisomer that correlates with the disease in a biological material from a healthy individual); or

disease state index value=[(a measurement value for an amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)/{(a measurement value for an amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)+(a measurement value for an enantiomer of the amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)}]÷[(a reference value for the amino acid stereoisomer that correlates with the disease from among reference values from a healthy individual)/{(a reference value for the amino acid stereoisomer that correlates with the disease from among reference values from the healthy individual)+(a reference value for the enantiomer of the amino acid stereoisomer that correlates with the disease from among reference values from the healthy individual)}].

In the method for diagnosing a disease according to the yet another aspect of the present invention, the step of outputting disease state information on the subject on the basis of the disease state index value may be a step of diagnosing that the subject is suffering from the disease when the disease state index value is 2.0 or greater.

In the method for diagnosing a disease according to the yet another aspect of the present invention, the amino acid stereoisomer that correlates with the disease may be: one, or two or more types of amino acids selected from the group consisting of D-serine, D-threonine, D-alanine, D-asparagine, allo-D-threonine, D-glutamine, D-proline and D-phenylalanine when the disease is a renal disease; D-histidine and/or D-asparagine when the disease is prostate gland cancer; or D-asparagine when the disease is osteoporosis.

The amino acid stereoisomer as referred to herein involves: 20 types of amino acids used in translation of proteins; 19 types of D-amino acids that are optical isomers of 19 types of L-amino acids, which are amino acids other than glycine; and allo-L-threonine, allo-D-threonine and allo-D-isoleucine.

The apparatus for analyzing a disease sample according to the aspect of the present invention includes: a member for measuring the amount of an amino acid stereoisomer in a biological material; a member for obtaining a disease state index value through a calculation by substituting the amount of the amino acid stereoisomer into a discriminant equation; and a member for outputting disease state information on the subject on the basis of the disease state index value. The member for measuring the amount of an amino acid stereoisomer in the biological material includes an automatic sample fractionation unit, and an HPLC separation and peak detection unit by way of a reverse phase column or the like. The member for obtaining an disease state index value through a calculation by substituting the amount of the amino acid stereoisomer into a discriminant equation includes: a memory unit for storing data such as the discriminant equation, reference values from healthy individuals for each of the disease, etc., and a computation unit for executing the calculation with the discriminant equation on the basis of the data. The member for outputting disease state information on the subject on the basis of the disease state index value includes: a disease state information selection unit; and a disease state information output unit. In addition thereto, the apparatus for analyzing a disease sample according to the aspect of the present invention may include: a control unit such as CPU for totally controlling the entire apparatus; an input and output interface unit for connecting an input device and an output device, and a communication interface unit for communicably connecting to a network.

The biological material as referred to herein involves: body fluids such as blood, plasma, serum, ascites, amniotic fluid, lymph fluid, saliva, seminal fluid and urine; excrement such as faeces, sweat and nasal discharge; and body tissues such as body hair, nail, skin tissues and visceral organ tissues, but not limited thereto.

The discriminant equation as referred to herein may involve a formula that calculates as to what times the measurement value of the amount of the amino acid stereoisomer in the subject is with respect to the reference value predetermined on the basis of a measurement value from a healthy individual. Also, the discriminant equation may involve a formula that calculates a proportion or a percentage of the amount of the amino acid stereoisomer with respect to a sum of the amount of the amino acid stereoisomer and the amount of enantiomer of the isomer. Furthermore, the discriminant equation may involve a formula that calculates a disease state index value from a combination of amounts of a plurality of types of amino acid stereoisomers. The plurality of amino acid stereoisomers may be a group of amino acids having a common feature of serving as a substrate for D-amino acid oxidase or D-aspartic acid oxidase, and the like. For example, among amino acid stereoisomers that serve as substrates of an identical enzyme, the amount of the amino acids correlating to a disease may be normalized with the amount of the amino acids not correlating to the disease.

In the discriminant equation according to the aspect of the present invention, a reference value for the amino acid stereoisomer that correlates with a disease in a biological material from a healthy individual is determined from an average or a median of the amounts of the amino acid stereoisomer that correlates with the disease in either one or both of biological materials from among a biological material from a healthy individual, and a biological material from other disease patient not correlating with the amino acid stereoisomer. Although the reference value may be predetermined, a case in which the reference value is a measurement value, or an average or a median thereof from a biological material prepared for and concomitantly tested on a control experiment in putting the present invention into practice is also acceptable.

As the disease state information according to the aspect of the present invention, outputting that “the subject is a healthy individual” is executed when the disease state index value calculated by the discriminant equation according to the aspect of the present invention is 1.0 or approximate thereto. When the disease state index value is 2.0 or greater, outputting that “the subject is highly probable of being suffering from the disease” may be executed. However, even when the disease state index value is less than 2.0, outputting that “the subject is probable of being suffering from the disease” may be executed.

The data on quantitative determination of the amino acid stereoisomer in a biological material from a subject obtained by the apparatus for analyzing a disease sample, the analysis system and the analysis method according to the aspects of the present invention can be used as an indicator for diagnoses and prophylaxes of a variety of diseases. In addition, the quantitative data can be used as an indicator for progression of the medical condition of the disease. Furthermore, the quantitative data may be used as an indicator for deciding the effectiveness of a medical drug for a treatment and/or prophylaxis of the disease. In addition, the quantitative data may be used as an indicator for deciding an influence of medical drugs, quasi-drugs, cosmetics, food and other chemical substances on living bodies, as well as influence of other physical and/or biological environmental factors on living bodies.

The entire contents of all documents referred to herein are incorporated herein by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 6 shows a Table summarizing analysis results of D-amino acids on each initial sample from a healthy individual and various types of disease patients;

FIG. 7 shows a distribution chart presenting serum concentrations of D-serine in each disease sample;

FIG. 8 shows a distribution chart presenting serum concentrations of L-serine in each disease sample;

FIG. 9 shows a distribution chart presenting serum concentrations of D-threonine in each disease sample;

FIG. 10 shows a distribution chart presenting serum concentrations of L-threonine in each disease sample;

FIG. 11 shows a distribution chart presenting serum concentrations of D-alanine in each disease sample;

FIG. 12 shows a distribution chart presenting serum concentrations of L-alanine in each disease sample;

FIG. 13 shows a distribution chart presenting percentages of the amount of the D-form of serine (% D) with respect to the sum of the amounts of the D-form and L-form of serine in samples;

FIG. 14 shows a distribution chart presenting percentages of the amount of the D-form of threonine (% D) with respect to the sum of the amounts of the D-form and L-form of threonine in samples;

FIG. 15 shows a distribution chart presenting percentages of the amount of the D-form of alanine (% D) with respect to the sum of the amounts of the D-form and L-form of alanine in samples;

FIG. 16 shows a bar chart presenting averages and standard errors of D-serine concentrations in the urine from three dilated cardiomyopathy model mice (MLP-KO mouse, hereinafter, may be referred to as “diseased”) and four control normal mice (hereinafter, may be referred to as “normal”). The ordinate indicates the D-serine concentration (nanomol/mL);

FIG. 17 shows a bar chart presenting averages and standard errors of L-serine concentrations in the urine from three dilated cardiomyopathy model mice (hereinafter, may be referred to as “diseased”) and four control normal mice (hereinafter, may be referred to as “normal”). The ordinate indicates the L-serine concentration (nanomol/mL);

FIG. 18 shows a bar chart presenting averages and standard errors of total serine concentrations (the sums of D-serine concentration and L-serine concentration) in the urine from three dilated cardiomyopathy model mice (hereinafter, may be referred to as “diseased”) and four control normal mice (hereinafter, may be referred to as “normal”). The ordinate indicates the total serine concentration (nanomol/mL);

FIG. 19 shows a bar chart presenting averages and standard errors of the percentages of D-serine concentration (% D) with respect to the total serine concentration in the urine from three dilated cardiomyopathy model mice (hereinafter, may be referred to as “diseased”) and four control normal mice (hereinafter, may be referred to as “normal”). The ordinate indicates the % D. In regard to the significant difference between the normal and the diseased, P was less than 0.02 according to a Student's t-test;

FIG. 20 shows a bar chart presenting averages and standard errors of D-arginine concentrations in the urine from three dilated cardiomyopathy model mice (hereinafter, may be referred to as “diseased”) and four control normal mice (hereinafter, may be referred to as “normal”). The ordinate indicates the D-arginine concentration (nanomol/mL);

FIG. 21 shows a bar chart presenting averages and standard errors of L-arginine concentrations in the urine from three dilated cardiomyopathy model mice (hereinafter, may be referred to as “diseased”) and four control normal mice (hereinafter, may be referred to as “normal”). The ordinate indicates the L-arginine concentration (nanomol/mL). In regard to the significant difference between the normal and the diseased, P was less than 0.01 according to a Student's t-test;

FIG. 22 shows a bar chart presenting averages and standard errors of total arginine concentrations (the sums of D-arginine concentration and L-arginine concentration) in the urine from three dilated cardiomyopathy model mice (hereinafter, may be referred to as “diseased”) and four control normal mice (hereinafter, may be referred to as “normal”). The ordinate indicates the total arginine concentration (nanomol/mL);

FIG. 23 shows a bar chart presenting averages and standard errors of D-glutamic acid concentrations in the urine from three dilated cardiomyopathy model mice (hereinafter, may be referred to as “diseased”) and four control normal mice (hereinafter, may be referred to as “normal”). The ordinate indicates the D-glutamic acid concentration (nanomol/mL). In regard to the significant difference between the normal and the diseased, P was less than 0.02 according to a Student's t-test;

FIG. 24 shows a bar chart presenting averages and standard errors of the L-glutamic acid concentrations in the urine from three dilated cardiomyopathy model mice (hereinafter, may be referred to as “diseased”) and four control normal mice (hereinafter, may be referred to as “normal”). The ordinate indicates the L-glutamic acid concentration (nanomol/mL);

FIG. 25 shows a bar chart presenting averages and standard errors of total glutamic acid concentrations (the sums of D-glutamic acid concentration and L-glutamic acid concentration) in the urine from three dilated cardiomyopathy model mice (hereinafter, may be referred to as “diseased”) and four control normal mice (hereinafter, may be referred to as “normal”). The ordinate indicates the total glutamic acid concentration (nanomol/mL);

FIG. 26 shows a bar chart presenting averages and standard errors of D-proline concentrations in the urine from three dilated cardiomyopathy model mice (hereinafter, may be referred to as “diseased”) and four control normal mice (hereinafter, may be referred to as “normal”). The ordinate indicates the D-proline concentration (nanomol/mL). In regard to the significant difference between the normal and the diseased, P was less than 0.01 according to a Student's t-test;

FIG. 27 shows a bar chart presenting averages and standard errors of L-proline concentrations in the urine from three dilated cardiomyopathy model mice (hereinafter, may be referred to as “diseased”) and four control normal mice (hereinafter, may be referred to as “normal”). The ordinate indicates the L-proline concentration (nanomol/mL);

FIG. 28 shows a bar chart presenting averages and standard errors of total proline concentrations (the sums of D-proline concentration and L-proline concentration) in the urine from three dilated cardiomyopathy model mice (hereinafter, may be referred to as “diseased”) and four control normal mice (hereinafter, may be referred to as “normal”). The ordinate indicates the total proline concentration (nanomol/mL);

FIG. 29 shows a bar chart presenting averages and standard errors of D-lysine concentrations in the urine from three dilated cardiomyopathy model mice (hereinafter, may be referred to as “diseased”) and four control normal mice (hereinafter, may be referred to as “normal”). The ordinate indicates the D-lysine concentration (nanomol/mL). In regard to the significant difference between the normal and the diseased, P was less than 0.01 according to a Student's t-test;

FIG. 30 shows a bar chart presenting averages and standard errors of L-lysine concentrations in the urine from three dilated cardiomyopathy model mice (hereinafter, may be referred to as “diseased”) and four control normal mice (hereinafter, may be referred to as “normal”). The ordinate indicates the L-lysine concentration (nanomol/mL);

FIG. 31 shows a bar chart presenting averages and standard errors of total lysine concentrations (the sums of D-lysine concentration and L-lysine concentration) in the urine from three dilated cardiomyopathy model mice (hereinafter, may be referred to as “diseased”) and four control normal mice (hereinafter, may be referred to as “normal”). The ordinate indicates the total lysine concentration (nanomol/mL);

FIG. 32 shows a graph presenting the change of averages and standard deviations of the body weights of 12 climacterium model mice (hereinafter, may be referred to as “OVX”) obtained by extirpation of the ovary from 9 weeks old HR-1 mice, and six control mice (hereinafter, may be referred to as “sham”) subjected to only skin incision and suture without carrying out the extirpation of the ovary from female mice of the same weeks old. The ordinate indicates the body weight (gram) of the mice. The black solid bars and the diagonally hatched bars show the average and standard deviation of the body weights of the mice before the operation (9 weeks old) and one week (10 weeks old), two weeks (11 weeks old), three weeks (12 weeks old) and four weeks (13 weeks old) after the operation of both climacterium model mice (OVX) and control mice (sham), respectively. In regard to the significant difference of the body weights between the climacterium model mice (OVX) and the control mice (sham), P was less than 0.05 (*) or less than 0.01 (**) according to a Student's t-test. In climacterium model mice obtained by extirpation of the ovary, the body weight significantly increased compared with the control mice from two weeks after the operation, suggesting that the climacterium model mice were successfully produced;

FIG. 33 shows a Table presenting results of the total analysis of amino acid optical isomer contents in the urine from the first mouse (OVX-3) among three climacterium model mice (OVX). Since no optical isomer of glycine is present, the results on glycine are described in a column of “L-form”. All amino acids were fluorescent-derivatized with NBD-F, and analyzed using the apparatus for the total analysis of amino acid optical isomer contents of the embodiment of the present invention. Since the NBD derivative of tryptophan is poorly sensitive, “nd” was designated in this analysis. Since cysteine produces cystine by air oxidization, the content of cysteine is extremely low, and thus “nd” was designated in this analysis;

FIG. 34 shows a Table presenting results of the total analysis of amino acid optical isomer contents in the urine from the second mouse (OVX-4) among three climacterium model mice (OVX). Since no optical isomer of glycine is present, the results on glycine are described in a column of “L-form”. All amino acids were fluorescent-derivatized with NBD-F, and analyzed using the apparatus for the total analysis of amino acid optical isomer contents of the embodiment of the present invention. Since the NBD derivative of tryptophan is poorly sensitive, “nd” was designated in this analysis. Since cysteine produces cystine by air oxidization, the content of cysteine is extremely low, and thus “nd” was designated in this analysis;

FIG. 35 shows a Table presenting results of the total analysis of amino acid optical isomer contents in the urine from the third mouse (OVX-5) among three climacterium model mice (OVX). Since no optical isomer of glycine is present, the results on glycine are described in a column of “L-form”. All amino acids were fluorescent-derivatized with NBD-F, and analyzed using the apparatus for the total analysis of amino acid optical isomer contents of the embodiment of the present invention. Since the NBD derivative of tryptophan is poorly sensitive, “nd” was designated in this analysis. Since cysteine produces cystine by air oxidization, the content of cysteine is extremely low, and thus “nd” was designated in this analysis;

FIG. 36 shows a Table presenting results of the total analysis of amino acid optical isomer contents in the urine from the first mouse (Sham-16) among four control mice (sham). Since no optical isomer of glycine is present, the results on glycine are described in a column of “L-form”. All amino acids were fluorescent-derivatized with NBD-F, and analyzed using the apparatus for the total analysis of amino acid optical isomer contents of the embodiment of the present invention. Since the NBD derivative of tryptophan is poorly sensitive, “nd” was designated in this analysis. Since cysteine produces cystine by air oxidization, the content of cysteine is extremely low, and thus “nd” was designated in this analysis;

FIG. 37 shows a Table presenting results of the total analysis of amino acid optical isomer contents in the urine from the second mouse (Sham-17) among four control mice (sham). Since no optical isomer of glycine is present, the results on glycine are described in a column of “L-form”. All amino acids were fluorescent-derivatized with NBD-F, and analyzed using the apparatus for the total analysis of amino acid optical isomer contents of the embodiment of the present invention. Since the NBD derivative of tryptophan is poorly sensitive, “nd” was designated in this analysis. Since cysteine produces cystine by air oxidization, the content of cysteine is extremely low, and thus “nd” was designated in this analysis;

FIG. 38 shows a Table presenting results of the total analysis of amino acid optical isomer contents in the urine from the third mouse (Sham-19) among four control mice (sham). Since no optical isomer of glycine is present, the results on glycine are described in a column of “L-form”. All amino acids were fluorescent-derivatized with NBD-F, and analyzed using the apparatus for the total analysis of amino acid optical isomer contents of the embodiment of the present invention. Since the NBD derivative of tryptophan is poorly sensitive, “nd” was designated in this analysis. Since cysteine produces cystine by air oxidization, the content of cysteine is extremely low, and thus “nd” was designated in this analysis;

FIG. 39 shows a Table presenting results of the total analysis of amino acid optical isomer contents in the urine from the fourth mouse (Sham-20) among four control mice (sham). Since no optical isomer of glycine is present, the results on glycine are described in a column of “L-form”. All amino acids were fluorescent-derivatized with NBD-F, and analyzed using the apparatus for the total analysis of amino acid optical isomer contents of the embodiment of the present invention. Since the NBD derivative of tryptophan is poorly sensitive, “nd” was designated in this analysis. Since cysteine produces cystine by air oxidization, the content of cysteine is extremely low, and thus “nd” was designated in this analysis;

FIG. 40 shows a Table comparing D-amino acid contents in the urine from three climacterium model mice (OVX) and four control mice (sham). The content of D-aspartic acid was lower in the control mice (sham) than in the climacterium model mice (OVX), and in regard to the significant difference, P was 0.015 according to a Student's t-test;

FIG. 41 shows a Table comparing L-amino acid contents in the urine from three climacterium model mice (OVX) and four control mice (sham). The contents of L-histidine and L-phenylalanine were lower in the climacterium model mice (OVX) than in the control mice (sham), and in regard to the significant difference, P was 0.017 and 0.037, respectively, according to a Student's t-test;

FIG. 42 shows a Table comparing percentages of the D-form concentration (% D) with respect to the sum of the D-form concentration and the L-form concentration of each amino acid in the urine from three climacterium model mice (OVX) and four control mice (sham). Since D-forms of threonine and isoleucine turn to be allo-forms in a living body, the % D was evaluated in terms of the percentage of the D-allo-form concentration with respect to the sum of the D-allo-form concentration and the L-form concentration. The percentage of D-aspartic acid was lower in the climacterium model mice (OVX) than in the control mice (sham), and in regard to the significant difference, P was 0.002 according to a Student's t-test;

FIG. 43 shows a Table comparing percentages of each D-amino acid concentration (% D/total L) with respect to the sum of the concentrations of all the L-amino acids in the urine from three climacterium model mice (OVX) and four control mice (sham). The percentage of D-aspartic acid was lower in the climacterium model mice (OVX) than in the control mice (sham), and in regard to the significant difference, P was 0.005 according to a Student's t-test;

FIG. 44 shows a Table comparing percentages of each D-amino acid concentration (% D/total D) with respect to the sum of the concentrations of all the D-amino acids in the urine from three climacterium model mice (OVX) and four control mice (sham). The percentage of D-aspartic acid was lower in the climacterium model mice (OVX) than in the control mice (sham), and in regard to the significant difference, P was 0.017 according to a Student's t-test;

FIG. 45 shows a Table comparing percentages of each D-amino acid concentration with respect to the D-glutamic acid concentration (% D/D-Glu) in the urine from three climacterium model mice (OVX) and four control mice (sham). Since D-aspartic acid is an acidic D-amino acid, an evaluation was made after a correction with the concentration of D-glutamic acid which is considered to be similarly metabolized. Also in the case of % D/D-Glu, the percentage of D-aspartic acid was lower in the climacterium model mice (OVX) than in the control mice (sham), and in regard to the significant difference, P was 0.006 according to a Student's t-test;

FIG. 46 shows a Table presenting results of the total analysis of amino acid optical isomer contents in the urine from the first mouse (S3) among three 6 weeks old male ICR mice transplanted with 2×107 sarcoma cells, in which growth of the explanted tumor was verified three weeks after the transplantation. Since no optical isomer of glycine is present, the results on glycine are described in a column of “L-form”. All amino acids were fluorescent-derivatized with NBD-F, and analyzed using the apparatus for the total analysis of amino acid optical isomer contents of the embodiment of the present invention. Since the NBD derivative of tryptophan is poorly sensitive, “nd” was designated in this analysis. Since cysteine produces cystine by air oxidization, the content of cysteine is extremely low, and thus “nd” was designated in this analysis;

FIG. 47 shows a Table presenting results of the total analysis of amino acid optical isomer contents in the urine from the second mouse (S4) among three 6 weeks old male ICR mice transplanted with 2×107 sarcoma cells, in which growth of the explanted tumor was verified three weeks after the transplantation. Since no optical isomer of glycine is present, the results on glycine are described in a column of “L-form”. All amino acids were fluorescent-derivatized with NBD-F, and analyzed using the apparatus for the total analysis of amino acid optical isomer contents of the embodiment of the present invention. Since the NBD derivative of tryptophan is poorly sensitive, “nd” was designated in this analysis. Since cysteine produces cystine by air oxidization, the content of cysteine is extremely low, and thus “nd” was designated in this analysis;

FIG. 48 shows a Table presenting results of the total analysis of amino acid optical isomer contents in the urine from the third mouse (S5) among three 6 weeks old male ICR mice transplanted with 2×107 sarcoma cells, in which growth of the explanted tumor was verified three weeks after the transplantation. Since no optical isomer of glycine is present, the results on glycine are described in a column of “L-form”. All amino acids were fluorescent-derivatized with NBD-F, and analyzed using the apparatus for the total analysis of amino acid optical isomer contents of the embodiment of the present invention. Since the NBD derivative of tryptophan is poorly sensitive, “nd” was designated in this analysis. Since cysteine produces cystine by air oxidization, the content of cysteine is extremely low, and thus “nd” was designated in this analysis;

FIG. 49 shows a Table presenting results of the total analysis of amino acid optical isomer contents in the urine from the first mouse (C3) among three 9 weeks old male ICR mice fed for a control experiment in the same environment as that of the sarcoma-transplanted mice. Since no optical isomer of glycine is present, the results on glycine are described in a column of “L-form”. All amino acids were fluorescent-derivatized with NBD-F, and analyzed using the apparatus for the total analysis of amino acid optical isomer contents of the embodiment of the present invention. Since the NBD derivative of tryptophan is poorly sensitive, “nd” was designated in this analysis. Since cysteine produces cystine by air oxidization, the content of cysteine is extremely low, and thus “nd” was designated in this analysis;

FIG. 50 shows a Table presenting results of the total analysis of amino acid optical isomer contents in the urine from the second mouse (C4) among three 9 weeks old male ICR mice fed for a control experiment in the same environment as that of the sarcoma-transplanted mice. Since no optical isomer of glycine is present, the results on glycine are described in a column of “L-form”. All amino acids were fluorescent-derivatized with NBD-F, and analyzed using the apparatus for the total analysis of amino acid optical isomer contents of the embodiment of the present invention. Since the NBD derivative of tryptophan is poorly sensitive, “nd” was designated in this analysis. Since cysteine produces cystine by air oxidization, the content of cysteine is extremely low, and thus “nd” was designated in this analysis;

FIG. 51 shows a Table presenting results of the total analysis of amino acid optical isomer contents in the urine from the third mouse (C5) among three 9 weeks old male ICR mice fed for a control experiment in the same environment as that of the sarcoma-transplanted mice. Since no optical isomer of glycine is present, the results on glycine are described in a column of “L-form”. All amino acids were fluorescent-derivatized with NBD-F, and analyzed using the apparatus for the total analysis of amino acid optical isomer contents of the embodiment of the present invention. Since the NBD derivative of tryptophan is poorly sensitive, “nd” was designated in this analysis. Since cysteine produces cystine by air oxidization, the content of cysteine is extremely low, and thus “nd” was designated in this analysis;

FIG. 52 shows a Table comparing D-amino acid contents in the urine from three sarcoma-transplanted mice (S3, S4 and S5) and three control mice (C3, C4 and C5). The content of D-arginine was higher in the sarcoma-transplanted mice than in the control mice, and in regard to the significant difference, P was 0.008 according to a Student's t-test;

FIG. 53 shows a Table comparing L-amino acid contents in the urine from three sarcoma-transplanted mice (S3, S4 and S5) and three control mice (C3, C4 and C5);

FIG. 54 shows a Table comparing percentages of the D-form concentration (% D) with respect to the sum of the D-form concentration and the L-form concentration of each amino acid in the urine from three sarcoma-transplanted mice (S3, S4 and S5) and three control mice (C3, C4 and C5). The % D of serine was lower in the sarcoma-transplanted mice than in the control mice, and in regard to the significant difference, P was 0.016 according to a Student's t-test;

FIG. 55 shows a Table comparing percentages of each D-amino acid concentration (% D/total L) with respect to the sum of the concentrations of all the L-amino acids in the urine from three sarcoma-transplanted mice (S3, S4 and S5) and three control mice (C3, C4 and C5);

FIG. 56 shows a Table comparing percentages of each D-amino acid concentration (% D/total D) with respect to the sum of the concentrations of all the D-amino acids in the urine from three sarcoma-transplanted mice (S3, S4 and S5) and three control mice (C3, C4 and C5). The percentages of D-asparagine and D-arginine tended to be higher in the sarcoma-transplanted mice than in the control mice, and in regard to the significant difference of D-arginine, P was 0.035 according to a Student's t-test;

FIG. 57 shows a Table comparing percentages of each D-amino acid concentration with respect to the D-asparagine concentration (% D/D-Asn) in the urine from three sarcoma-transplanted mice (S3, S4 and S5) and three control mice (C3, C4 and C5). D-asparagine was used as a basis for a correction of the concentration in the urine since D-asparagine was present in mammalian urine at a comparatively high concentration, and the proportion with respect to the total D-amino acid concentration was most stable. The percentage of D-arginine tended to be higher in the sarcoma-transplanted mice than in the control mice, and in regard to the significant difference, P was 0.016 according to a Student's t-test;

FIG. 58 shows a bar chart presenting averages and standard errors of D-serine concentrations (D-Ser), L-serine concentrations (L-Ser) and the sum of both concentrations (Ser) in the urine from three Alzheimer's disease model mice (8 weeks old male hemizygote mice of an amyloid β precursor protein-highly expressing transgenic mouse Tg2576, hereinafter, may be referred to as “hemi”) and three control normal mice (C57BL, hereinafter, may be referred to as “Wild”). The ordinate indicates the concentration (nanomol/mL);

FIG. 59 shows a bar chart presenting averages and standard errors of the percentages of D-serine concentration (% D) with respect to the total serine concentration in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). The ordinate indicates the % D;

FIG. 60 shows a graph presenting a relative ratio of the D-serine concentration to the D-allo-threonine concentration (D-Ser/D-allo-Thr) or a relative ratio of the D-serine concentration to the D-allo-isoleucine concentration (D-Ser/D-allo-Ile) in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). Both the relative ratio of the D-serine concentration to the D-allo-threonine concentration in the urine, and the relative ratio of the D-serine concentration to the D-allo-isoleucine concentration in the urine were higher in the Alzheimer's disease model mice than in the control mice, and in regard to the significant difference, P was less than 0.01 according to a Student's t-test in both cases;

FIG. 61 shows a bar chart presenting averages and standard errors of D-alanine concentrations (D-Ala), L-alanine concentrations (L-Ala) and the sum of both concentrations (Ala) in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). The ordinate indicates the concentration (nanomol/mL). The D-alanine concentration in the urine was higher in the Alzheimer's disease model mice than in the control normal mice, and in regard to the significant difference, P was less than 0.01 according to a Student's t-test;

FIG. 62 shows a bar chart presenting averages and standard errors of the percentages of D-alanine concentration (% D) with respect to the total alanine concentration in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). The ordinate indicates the % D. The percentage of the D-alanine concentration (% D) with respect to the total alanine concentration in the urine was higher in the Alzheimer's disease model mice than in the control normal mice, and in regard to the significant difference, P was less than 0.01 according to a Student's t-test;

FIG. 63 shows a bar chart presenting averages and standard errors of D-methionine concentrations (D-Met), L-methionine concentrations (L-Met) and the sum of both concentrations (Met) in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). The ordinate indicates the concentration (nanomol/mL). The D-Met concentration in the urine tended to be higher in the Alzheimer's disease model mice than in the control normal mice;

FIG. 64 shows a bar chart presenting averages and standard errors of the percentages of D-methionine concentration (% D) with respect to the total serine concentration in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). The ordinate indicates the % D;

FIG. 65 shows a graph presenting a relative ratio of the D-methionine concentration to the D-allo-threonine concentration (D-Met/D-allo-Thr) or a relative ratio of the D-methionine concentration to the D-allo-isoleucine concentration (D-Met/D-allo-Ile) in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). Both the relative ratio of the D-methionine concentration to the D-allo-threonine concentration in the urine, and the relative ratio of the D-methionine concentration to the D-allo-isoleucine concentration in the urine were higher in the Alzheimer's disease model mice than in the control mice, and in regard to the significant difference, P was less than 0.05 according to a Student's t-test in both cases;

FIG. 66 shows a bar chart presenting averages and standard errors of D-leucine concentrations (D-Leu), L-leucine concentrations (L-Leu) and the sum of both concentrations (Leu) in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). The ordinate indicates the concentration (nanomol/mL);

FIG. 67 shows a bar chart presenting averages and standard errors of the percentages of D-leucine concentration (% D) with respect to the total serine concentration in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). The ordinate indicates the % D;

FIG. 68 shows a graph presenting a relative ratio of the D-leucine concentration to the D-allo-threonine concentration (D-Leu/D-allo-Thr) or a relative ratio of the D-leucine concentration to the D-allo-isoleucine concentration (D-Leu/D-allo-Ile) in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). Both the relative ratio of the D-leucine concentration to the D-allo-threonine concentration in the urine, and the relative ratio of the D-leucine concentration to the D-allo-isoleucine concentration in the urine were higher in the Alzheimer's disease model mice than in the control mice, and in regard to the significant difference, P was less than 0.05 and less than 0.01, respectively, according to a Student's t-test;

FIG. 69 shows a bar chart presenting averages and standard errors of D-aspartic acid concentrations (D-Asp), L-aspartic acid concentrations (L-Asp) and the sum of both concentrations (Asp) in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). The ordinate indicates the concentration (nanomol/mL). The D-aspartic acid concentration in the urine was lower in the Alzheimer's disease model mice than in the control normal mice, and in regard to the significant difference, P was less than 0.05 according to a Student's t-test;

FIG. 70 shows a bar chart presenting averages and standard errors of the percentages of the D-aspartic acid concentration (% D) with respect to the total aspartic acid concentration in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). The ordinate indicates the % D;

FIG. 71 shows a bar chart presenting averages and standard errors of D-phenylalanine concentrations (D-Phe), L-phenylalanine concentrations (L-Phe) and the sum of both concentrations (Phe) in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). The ordinate indicates the concentration (nanomol/mL);

FIG. 72 shows a bar chart presenting averages and standard errors of the percentages of D-phenylalanine concentration (% D) with respect to the total phenylalanine concentration in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). The ordinate indicates the % D;

FIG. 73 shows a graph presenting a relative ratio of the D-phenylalanine concentration to the D-allo-threonine concentration (D-Phe/D-allo-Thr) or a relative ratio of the D-phenylalanine concentration to the D-allo-isoleucine concentration (D-Phe/D-allo-Ile) in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). The relative ratio of the D-phenylalanine concentration to the D-allo-isoleucine concentration in the urine was higher in the Alzheimer's disease model mice than in the control mice, and in regard to the significant difference, P was less than 0.05 according to a Student's t-test;

FIG. 74 shows a Table presenting results of the total analysis of amino acid optical isomer contents in the urine from the first mouse (DAO+/+1) among three D-amino acid oxidase (DAO) wild type mice (ddY/DAO+, which may be referred to as “DAO+/+”). Since no optical isomer of glycine is present, the results on glycine are described in a column of “L-form”. All amino acids were fluorescent-derivatized with NBD-F, and analyzed using the apparatus for the total analysis of amino acid optical isomer contents of the embodiment of the present invention. Since the NBD derivative of tryptophan is poorly sensitive, “nd” was designated in this analysis. Since cysteine produces cystine by air oxidization, the content of cysteine is extremely low, and thus “nd” was designated in this analysis;

FIG. 75 shows a Table presenting results of the total analysis of amino acid optical isomer contents in the urine from the second mouse (DAO+/+2) among three D-amino acid oxidase (DAO) wild type mice (ddY/DAO+, which may be referred to as “DAO+/+”). Since no optical isomer of glycine is present, the results on glycine are described in a column of “L-form”. All amino acids were fluorescent-derivatized with NBD-F, and analyzed using the apparatus for the total analysis of amino acid optical isomer contents of the embodiment of the present invention. Since the NBD derivative of tryptophan is poorly sensitive, “nd” was designated in this analysis. Since cysteine produces cystine by air oxidization, the content of cysteine is extremely low, and thus “nd” was designated in this analysis;

FIG. 76 shows a Table presenting results of the total analysis of amino acid optical isomer contents in the urine from the third mouse (DAO+/+3) among three D-amino acid oxidase (DAO) wild type mice (ddY/DAO+, which may be referred to as “DAO+/+”). Since no optical isomer of glycine is present, the results on glycine are described in a column of “L-form”. All amino acids were fluorescent-derivatized with NBD-F, and analyzed using the apparatus for the total analysis of amino acid optical isomer contents of the embodiment of the present invention. Since the NBD derivative of tryptophan is poorly sensitive, “nd” was designated in this analysis. Since cysteine produces cystine by air oxidization, the content of cysteine is extremely low, and thus “nd” was designated in this analysis;

FIG. 77 shows a Table presenting results of the total analysis of amino acid optical isomer contents in the urine from the first mouse (DAO−/−1) among three D-amino acid oxidase (DAO) deficient mice (ddY/DAO−, which may be referred to as “DAO−/−”). Since no optical isomer of glycine is present, the results on glycine are described in a column of “L-form”. All amino acids were fluorescent-derivatized with NBD-F, and analyzed using the apparatus for the total analysis of amino acid optical isomer contents of the embodiment of the present invention. Since the NBD derivative of tryptophan is poorly sensitive, “nd” was designated in this analysis. Since cysteine produces cystine by air oxidization, the content of cysteine is extremely low, and thus “nd” was designated in this analysis;

FIG. 78 shows a Table presenting results of the total analysis of amino acid optical isomer contents in the urine from the second mouse (DAO−/−2) among three D-amino acid oxidase (DAO) deficient mice (ddY/DAO−, which may be referred to as “DAO−/−”). Since no optical isomer of glycine is present, the results on glycine are described in a column of “L-form”. All amino acids were fluorescent-derivatized with NBD-F, and analyzed using the apparatus for the total analysis of amino acid optical isomer contents of the embodiment of the present invention. Since the NBD derivative of tryptophan is poorly sensitive, “nd” was designated in this analysis. Since cysteine produces cystine by air oxidization, the content of cysteine is extremely low, and thus “nd” was designated in this analysis;

FIG. 79 shows a Table presenting results of the total analysis of amino acid optical isomer contents in the urine from the third mouse (DAO−/−3) among three D-amino acid oxidase (DAO) deficient mice (ddY/DAO−, which may be referred to as “DAO−/−”). Since no optical isomer of glycine is present, the results on glycine are described in a column of “L-form”. All amino acids were fluorescent-derivatized with NBD-F, and analyzed using the apparatus for the total analysis of amino acid optical isomer contents of the embodiment of the present invention. Since the NBD derivative of tryptophan is poorly sensitive, “nd” was designated in this analysis. Since cysteine produces cystine by air oxidization, the content of cysteine is extremely low, and thus “nd” was designated in this analysis;

FIG. 80 shows a Table comparing D-amino acid contents in the urine from three D-amino acid oxidase (DAO) wild type mice (DAO+/+1, DAO+/+2 and DAO+/+3) and three D-amino acid oxidase (DAO) deficient mice (DAO−/−1, DAO−/−2 and DAO−/−3). The contents of D-serine, D-allo-threonine, D-alanine, D-proline, D-leucine and D-phenylalanine were significantly higher in the D-amino acid oxidase (DAO) enzyme deficient mice than in the DAO wild type mice;

FIG. 81 shows a Table comparing L-amino acid contents in the urine from three D-amino acid oxidase (DAO) wild type mice (DAO+/+1, DAO+/+2 and DAO+/+3) and three D-amino acid oxidase (DAO) deficient mice (DAO−/−1, DAO−/−2 and DAO−/−3);

FIG. 82 shows a Table comparing percentages of the D-form concentration (% D) with respect to the sum of the D-form concentration and the L-form concentration of each amino acid in the urine from three D-amino acid oxidase (DAO) wild type mice (DAO+/+1, DAO+/+2 and DAO+/+3) and three D-amino acid oxidase (DAO) deficient mice (DAO−/−1, DAO−/−2 and DAO−/−3). The percentages of D-serine, D-allo-threonine, D-alanine, D-proline, D-leucine and D-phenylalanine were significantly higher in the D-amino acid oxidase (DAO) enzyme deficient mice than in the DAO wild type mice;

FIG. 83 shows a Table comparing percentages of each D-amino acid concentration (% D/total L) with respect to the sum of the concentrations of all the L-amino acids in the urine from three D-amino acid oxidase (DAO) wild type mice (DAO+/+1, DAO+/+2 and DAO+/+3) and three D-amino acid oxidase (DAO) deficient mice (DAO−/−1, DAO−/−2 and DAO−/−3). The percentages of D-serine, D-allo-threonine, D-alanine, D-leucine and D-phenylalanine were significantly higher in the D-amino acid oxidase (DAO) enzyme deficient mice than in the DAO wild type mice;

FIG. 84 shows a Table comparing percentages of each D-amino acid concentration (% D/total D) with respect to the sum of the concentrations of all the D-amino acids in the urine from three D-amino acid oxidase (DAO) wild type mice (DAO+/+1, DAO+/+2 and DAO+/+3) and three D-amino acid oxidase (DAO) deficient mice (DAO−/−1, DAO−/−2 and DAO−/−3);

FIG. 85 shows a Table comparing percentages of each D-amino acid concentration (% D/D-Asn) with respect to the D-asparagine concentration in the urine from three D-amino acid oxidase (DAO) wild type mice (DAO+/+1, DAO+/+2 and DAO+/+3) and three D-amino acid oxidase (DAO) deficient mice (DAO−/−1, DAO−/−2 and DAO−/−3). The percentages of D-serine, D-allo-threonine, D-alanine, D-proline, D-leucine and D-phenylalanine were significantly higher in the D-amino acid oxidase (DAO) enzyme deficient mice than in the DAO wild type mice;

FIG. 86 shows a wave form chart presenting results of the total analysis of amino acid optical isomer contents in the urine from the first mouse among four D-aspartate oxidase (DDO) wild type mice (DDO+);

FIG. 87 shows a wave form chart presenting results of the total analysis of amino acid optical isomer contents in the urine from the second mouse among four D-aspartate oxidase (DDO) wild type mice (DDO+);

FIG. 88 shows a wave form chart presenting results of the total analysis of amino acid optical isomer contents in the urine from the third mouse among four D-aspartate oxidase (DDO) wild type mice (DDO+);

FIG. 89 shows a wave form chart presenting results of the total analysis of amino acid optical isomer contents in the urine from the fourth mouse among four D-aspartate oxidase (DDO) wild type mice (DDO+);

FIG. 90 shows a wave form chart presenting results of the total analysis of amino acid optical isomer contents in the urine from the first mouse among four D-aspartate oxidase (DDO) deficient mice (DDO−);

FIG. 91 shows a wave form chart presenting results of the total analysis of amino acid optical isomer contents in the urine from the second mouse among four D-aspartate oxidase (DDO) deficient mice (DDO−);

FIG. 92 shows a wave form chart presenting results of the total analysis of amino acid optical isomer contents in the urine from the third mouse among four D-aspartate oxidase (DDO) deficient mice (DDO−);

FIG. 93 shows a wave form chart presenting results of the total analysis of amino acid optical isomer contents in the urine from the fourth mouse among four D-aspartate oxidase (DDO) deficient mice (DDO−);

FIG. 94 shows a bar chart presenting D-valine concentrations (nanomol/mL) and standard errors thereof in the plasma from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid);

FIG. 95 shows a bar chart presenting D-allo-isoleucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid);

FIG. 96 shows a bar chart presenting D-isoleucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid);

FIG. 97 shows a bar chart presenting D-leucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid);

FIG. 98 shows a bar chart presenting D-phenylalanine concentrations (nanomol/mL) and standard errors thereof in the plasma from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid). In regard to the significant difference, p is less than 0.01;

FIG. 99 shows a bar chart presenting L-valine concentrations (nanomol/mL) and standard errors thereof in the plasma from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid);

FIG. 100 shows a bar chart presenting L-allo-isoleucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid);

FIG. 101 shows a bar chart presenting L-isoleucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid);

FIG. 102 shows a bar chart presenting L-leucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid);

FIG. 103 shows a bar chart presenting L-phenylalanine concentrations (nanomol/mL) and standard errors thereof in the plasma from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid). In regard to the significant difference, p is less than 0.01;

FIG. 104 shows a bar chart presenting D-valine concentrations (nanomol/mL) and standard errors thereof in the urine from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid);

FIG. 105 shows a bar chart presenting D-allo-isoleucine concentrations (nanomol/mL) and standard errors thereof in the urine from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid);

FIG. 106 shows a bar chart presenting D-isoleucine concentrations (nanomol/mL) and standard errors thereof in the urine from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid);

FIG. 107 shows a bar chart presenting D-leucine concentrations (nanomol/mL) and standard errors thereof in the urine from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid);

FIG. 108 shows a bar chart presenting D-phenylalanine concentrations (nanomol/mL) and standard errors thereof in the urine from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid);

FIG. 109 shows a bar chart presenting L-valine concentrations (nanomol/mL) and standard errors thereof in the urine from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid);

FIG. 110 shows a bar chart presenting L-allo-isoleucine concentrations (nanomol/mL) and standard errors thereof in the urine from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid);

FIG. 111 shows a bar chart presenting L-isoleucine concentrations (nanomol/mL) and standard errors thereof in the urine from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid);

FIG. 112 shows a bar chart presenting L-leucine concentrations (nanomol/mL) and standard errors thereof in the urine from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid);

FIG. 113 shows a bar chart presenting L-phenylalanine concentrations (nanomol/mL) and standard errors thereof in the urine from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid). In regard to the significant difference, p is less than 0.01;

FIG. 114 shows a bar chart presenting D-valine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid);

FIG. 115 shows a bar chart presenting D-allo-isoleucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid);

FIG. 116 shows a bar chart presenting D-isoleucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). In regard to the significant difference, p is less than 0.05;

FIG. 117 shows a bar chart presenting D-leucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid);

FIG. 118 shows a bar chart presenting D-phenylalanine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid);

FIG. 119 shows a bar chart presenting L-valine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). In regard to the significant difference, p is less than 0.01;

FIG. 120 shows a bar chart presenting L-allo-isoleucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). In regard to the significant difference, p is less than 0.01;

FIG. 121 shows a bar chart presenting L-isoleucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). In regard to the significant difference, p is less than 0.01;

FIG. 122 shows a bar chart presenting L-leucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). In regard to the significant difference, p is less than 0.05;

FIG. 123 shows a bar chart presenting L-phenylalanine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid);

FIG. 124 shows a bar chart presenting D-isoleucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease intermediate type mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, +/Dbt^(tmlgeh); grey solid) and five control mice (white solid). In regard to the significant difference, p is less than 0.01;

FIG. 125 shows a bar chart presenting L-valine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease intermediate type mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, +/Dbt^(tmlgeh); grey solid) and five control mice (white solid);

FIG. 126 shows a bar chart presenting L-allo-isoleucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease intermediate type mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, +/Dbt^(tmlgeh); grey solid) and five control mice (white solid);

FIG. 127 shows a bar chart presenting L-isoleucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease intermediate type mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, +/Dbt^(tmlgeh); grey solid) and five control mice (white solid);

FIG. 128 shows a bar chart presenting L-leucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease intermediate type mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, +/Dbt^(tmlgeh); grey solid) and five control mice (white solid);

FIG. 129 shows a bar chart presenting D-valine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease intermediate type mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid);

FIG. 130 shows a bar chart presenting D-allo-isoleucine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid);

FIG. 131 shows a bar chart presenting D-isoleucine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). In regard to the significant difference, p is less than 0.05;

FIG. 132 shows a bar chart presenting D-leucine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid);

FIG. 133 shows a bar chart presenting D-phenylalanine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid);

FIG. 134 shows a bar chart presenting L-valine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). In regard to the significant difference, p is less than 0.01;

FIG. 135 shows a bar chart presenting L-allo-isoleucine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). In regard to the significant difference, p is less than 0.01;

FIG. 136 shows a bar chart presenting L-isoleucine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). In regard to the significant difference, p is less than 0.05;

FIG. 137 shows a bar chart presenting L-leucine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). In regard to the significant difference, p is less than 0.01;

FIG. 138 shows a bar chart presenting L-phenylalanine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid);

FIG. 139 shows a bar chart presenting D-isoleucine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease intermediate type mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, +/Dbt^(tmlgeh); grey solid) and five control mice (white solid). In regard to the significant difference, p is less than 0.05;

FIG. 140 shows a bar chart presenting L-valine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease intermediate type mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, +/Dbt^(tmlgeh); grey solid) and five control mice (white solid);

FIG. 141 shows a bar chart presenting L-allo-isoleucine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease intermediate type mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, +/Dbt^(tmlgeh); grey solid) and five control mice (white solid);

FIG. 142 shows a bar chart presenting L-isoleucine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease intermediate type mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, +/Dbt^(tmlgeh); grey solid) and five control mice (white solid);

FIG. 143 shows a bar chart presenting L-leucine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease intermediate type mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, +/Dbt^(tmlgeh); grey solid) and five control mice (white solid);

FIG. 144 shows an additional Table summarizing analysis results of D-amino acids in each initial sample from various types of disease patients; and

FIG. 145 shows an additional Table summarizing analysis results of L-amino acids in each initial sample from various types of disease patients.

DESCRIPTION OF EMBODIMENTS

Examples of the invention described below have been presented for the purpose of illustration only, and are not to be construed as limiting the technical scope of the invention. The technical scope of the present invention is limited solely by the language of the appended claims. Modifications of the present invention such as, for example, additions, deletions and substitutions of features of the present invention may be made without departing from the spirit of the present invention.

The following Examples were conducted in accordance with guidelines from National Institutes of Health (NIH) after an approval by an ethics committee of Shiseido Research Center.

Example 1

Total Analysis of Amino Acid Stereoisomers in Samples Derived From Various Types of Disease Patients

(1)

1. Materials and Methods

(1) Sample Serum samples derived from healthy humans and various types of disease patients were obtained from BioServe Biotechnologies, Ltd., USA, Maryland, Beltsville). The samples were collected by Genomic Collaborative Inc., which was purchased by BioServe Biotechnologies, Ltd. Blood collection was carried out under a control by an attending physician of the patient, and a consent by the blood donor on a document in accordance with United States Health Insurance Portability and Accountability Act (HIPAA) was confirmed. Among the donors of the samples employed in the analyses, healthy individuals selected were four males and four females, all 50 years old Caucasian, not having the past history of the subject disease to be studied. Renal disease patients selected were not suffering from diabetes in combination. Cognitive impairment patients selected were four males who were diagnosed to fall under the severity of stage 5 (stage when necessity for assistance from someone for daily life has started) among 7 stages defined by Alzheimer's disease association. Large intestine cancer patients selected were four male who were diagnosed to fall under stage II. Breast cancer patients selected were four females who were diagnosed to fall under stage II, with the cancer developed on only one side. A Prostate gland cancer patient selected was a male who was diagnosed to fall under stage II, and not treated by any of hormone therapy and chemotherapy. A hepatopathy patient selected was a female who was diagnosed to fall under stage II, with hepatopathy developed on only one side. An osteoporosis patient selected was a female. An ovary cancer patient selected was a female falling under stage II. The donors of the samples used in the present analyses excluded smokers.

(2) Total Analysis of Amino Acid Stereoisomers

The samples were subjected to a total analysis of amino acid stereoisomers by a D, L-amino acid simultaneous and highly sensitive analysis system developed by Zaitsu et. al., (Japanese Patent No. 4291628), entitled LIQUID CHROMATOGRAPHY APPARATUS AND METHOD OF ANALYZING OPTICAL ISOMER CONTAINED IN SAMPLE. Details of analytical conditions of each amino acid are described in Hamase K. et el., J. Chromatogr. A, 1143: 105 (2007); Hamase K., et el., J. Chromatogr. A, 1217: 1056 (2010); and Miyoshi Y., et el., J. Chromatogr. B, 879: 3184 (2011). In brief, the sample was homogenized in 20 times volume of methanol at 4° C. and 3,500 rpm, for 2 min using a microhomogenizing system (Micro Smash MS-100R, Tomy Seiko Co., Ltd.), followed by centrifugation at 20,400×g for 10 min. The centrifugation supernatant in a volume of 10 μL was dried under reduced pressure at 40° C. To the resulting residue were added 20 μL of 200 mM sodium borate buffer (pH 8.0), and 5 μL of a 40 mM NBD-F (4-fluoro-7-nitro-2,1,3-benzooxadiazole, Tokyo Chemical Industry Co., Ltd.) solution in anhydrous methyl cyanide, and the mixture was heated at 60° C., for 2 min. After completion of the reaction, 75 μL of a 2% (v/v) aqueous trifluoroacetic acid solution was added thereto. This mixture in a volume of 2 μL was subjected to an HPLC system (NANOSPACE SI-2; Shiseido Co., Ltd., see supplementary information of Sasabe, J. et el., Proc. Natl. Acad. Sci., 109: 627 (2012)). In brief, an analytical column for reverse phase separation employed was a monolithic ODS column (in-house product; internal diameter: 759 mm×0.53 mm, attached to a quartz glass capillary) which had been incubated at 40° C. As a mobile phase, a mixture containing methyl cyanide, trifluoroacetic acid and water (volume ratio of methyl cyanide:trifluoroacetic acid:water=5:0.05:95) was used. The flow rate was 35 μL per minute. After the reverse phase separation, a NBD-modified amino acid fraction intended was automatically transferred to an enantio-selective column via a column switching valve to which a 150-μL loop was attached. For enantiomer separation, a SUMICHIRAL OA-2500S column in which (S)-naphthylglycine is used as a chiral selector (internal diameter: 250 mm×1.5 mm I.D., packed in-house, material: manufactured by Sumika Chemical Analysis Service, Ltd) was employed. Fluorescent detection was executed with an excitation wavelength of 470 nm and a detection wavelength of 530 nm.

2. Results

FIG. 1 shows a Table summarizing analysis results of D-amino acids on each initial samples from healthy individuals and various types of disease patients. Each line in the Table shows a disease name of the sample donor, and each row shows the type of D-amino acid analyzed. In Table, ND denotes “being the detection limit or below”. Coarse backward diagonal hatching patterns in rows of tryptophan, cysteine and tyrosine indicate that quantitative determination of these amino acids failed on the sample at issue. Upward arrowheads indicate that the sample contained the amino acid in an amount more than that in the samples from healthy males shown in the first line, whereas downward arrowheads indicate that the sample contained the amino acid in an amount less than that in the samples from the healthy males shown in the first line. Fine backward diagonal hatching patterns (for example, asparagine in renal disease samples, etc.) indicate that a ratio of the amount of the amino acid in these samples to the amount of the amino acid in the samples from the healthy males is two times or greater (upward arrowhead), or ½ or less (downward arrowhead). Coarse downward diagonal hatching patterns (for example, glutamine in renal disease samples, etc.) indicate that a ratio of the amount of the amino acid in these samples to the amount of the amino acid in the samples from the healthy males is less than two times (upward arrowhead), or less than ½ (downward arrowhead).

Among the D-amino acids the serum concentration of which was higher in the samples from renal disease patients than in the samples from healthy males shown in FIG. 6, serine, allo-D-threonine, threonine, alanine, proline and phenylalanine are substrates for D-amino acid oxidase; however, aspartic acid and glutamine are substrates for D-aspartate oxidase. In addition, serum concentrations of histidine, arginine, methionine, valine, allo-D-isoleucine and isoleucine were decided not to vary between the renal disease patients and the healthy males, even though these amino acids are substrates for the same D-amino acid oxidase. Furthermore, although aspartic acid and glutamic acid are substrates for D-aspartate oxidase, it was determined that there was no difference in the serum concentrations of these between the renal disease patients and the healthy males. In these regards, it is impossible to explain the higher serum concentrations of the aforementioned types of D-amino acids in the samples from renal disease patients as compared with the samples from healthy males, on the basis of only the specificity of metabolic enzyme of D-amino acids conventionally known. The D-amino acids the serum concentration of which was higher in the sample from the prostate gland cancer patient than in the samples from healthy males were D-histidine and D-asparagine, and the D-amino acid the serum concentration of which was higher in the sample from the osteoporosis patient than in the samples from healthy males was D-asparagine. Also in these regards, it is impossible to explain the higher serum concentrations of these D-amino acids, on the basis of only the specificity of metabolic enzyme of D-amino acids conventionally known. Both D-amino acid oxidase and D-aspartate oxidase have been known to be localized in proximal tubule of kidney. Thus, these enzyme activities in renal disease patients can be expected to decrease. In this instance, it would be considered that degradation of all D-amino acids that may be substrates of these enzymes is suppressed, whereby the amount thereof in the body increases. However, the amount of only part of the D-amino acids in the body, but not all of the D-amino acids, increases in fact, although the mechanism of this event is not known. It is suggested that a change of the serum concentration of the D-amino acid specific to the diseases can be utilized in the diagnosis for the disease.

FIG. 6 suggests that there is no difference in amount regarding the greater part of types of the D-amino acids between healthy males and various types of disease patients; however, the amount of some types of amino acids was two times or more or ½ or less as compared with that in healthy males. Accordingly, focus was first made to a renal disease in which a significant difference was found in comparatively many types of D-amino acids as compared with the samples from healthy males, and thus samples from four donors suffering from the same disease were studied in detail.

FIG. 7, FIG. 8, FIG. 9, FIG. 10, FIG. 11 and FIG. 12 show a distribution chart presenting serum concentrations of D-serine, L-serine, D-threonine, L-threonine, D-alanine and L-alanine, respectively, in each disease sample. The sample 1 was from healthy males; the sample 2 was from renal disease patients; the sample 3 was from cognitive impairment patients; the sample 4 was from healthy females; the sample 5 was from large intestine cancer patients; and the sample 6 was from breast cancer patients. FIG. 7 indicates that the serum concentrations of D-serine in healthy male and female individuals, and those in the cognitive impairment, large intestine cancer and breast cancer patients were almost constant and low, whereas the serum concentrations of D-serine in renal disease patients were higher than those in other samples by at least two times or more. FIG. 9 indicates that the serum concentrations of D-threonine in the healthy male and female individuals, and those in the cognitive impairment, large intestine cancer and breast cancer patients were almost constant and low, whereas the serum concentrations of D-threonine in renal disease patients were higher than those in other samples by at least two times or more. Similarly, FIG. 11 indicates that the serum concentrations of D-alanine in the healthy male and female individuals, and those in the cognitive impairment, large intestine cancer and breast cancer patients were almost constant and low, whereas the serum concentrations of D-alanine in three among four renal disease patients were higher than those in other samples by at least two times or more. To the contrary, FIG. 8, FIG. 10 and FIG. 12 indicate that with respect to any one of L-serine, L-threonine and L-alanine, expanded ranges of distribution of the amounts of the amino acids in healthy individuals, and four types of the disease patients studied in this Example were found due to the individual differences. Moreover, in the samples from the renal disease patients, such remarkable differences as was the case with D-serine, D-threonine and D-alanine were not found in total for L-serine, L-threonine and L-alanine, although the amounts thereof tend to be somewhat smaller than those in the samples from the healthy male and female individuals, and other disease patients.

A characteristic feature of the serum concentrations of D-amino acids clarified from FIG. 7 to FIG. 12 is that any serum concentration of the D-amino acids in the samples from the healthy male and female individuals was very low, with a small deviation. The same applies to other D-amino acids (data not shown). Furthermore, in the case in which a change of the serum concentrations of D-amino acids in the samples from the patients that correlates with the disease is not found, the serum concentration of any of the D-amino acids was very low, with a small deviation, similarly to the cases of healthy individuals. This feature leads to a lower frequency of false positive results in diagnoses, which presents an important basis for usability in diagnoses according to the amount of the D-amino acid. In addition, when the amount of a D-amino acid that varies specifically to a disease is represented by a parameter combined with other factor, as long as a correlation of the other factor per se with the disease is inferior to that of the amount of the D-amino acid, only a diagnostic characteristic equivalent to that of a diagnosis conducted on the basis only of the amount of the D-amino acid is substantially attained.

FIG. 13, FIG. 14 and FIG. 15 show a distribution chart presenting percentages of the amount of D-form (% D) with respect to the sum of the amounts of the D-form and L-form of serine, threonine and alanine in samples, respectively. FIG. 13 indicates that the percentages of the serum concentrations of D-serine with respect to the total serum concentration of serine in the healthy male and female individuals, and those in the cognitive impairment, large intestine cancer and breast cancer patients were almost constant and low, whereas the percentages of the serum concentrations of D-serine with respect to the total serum concentration of serine in renal disease patients were higher than those in other samples by 4 times or more. FIG. 14 indicates that the percentages of the serum concentrations of D-threonine with respect to the total serum concentration of threonine in the healthy male and female individuals, and those in the cognitive impairment, large intestine cancer and breast cancer patients were almost constant and low, whereas the percentages of the serum concentrations of D-threonine with respect to the total serum concentration of threonine in renal disease patients were higher than those in other samples by 4 times or more. Similarly, FIG. 15 indicates that the percentages of the serum concentrations of D-alanine with respect to the total serum concentration of alanine in the healthy male and female individuals, and those in the cognitive impairment, large intestine cancer and breast cancer patients were almost constant and low, whereas the percentages of the serum concentrations of D-alanine with respect to the total serum concentration of alanine in three among four renal disease patients were higher than those in other samples by 4 times or more.

Example 2

Total Analysis of Amino Acid Stereoisomers in Samples Derived from Mouse Disease Model

1. Materials and Methods

1.1 Total Analysis of Amino Acid Stereoisomers

For the Total analysis of amino acid stereoisomers, a system similar to the D, L-amino acid simultaneous and highly sensitive analysis system explained in Example 1 was used, except that MS not having a damper was used as a liquid feeding pump, and that selection of a secondary mobile phase from a wide range of options was enabled through adopting MPS and a low-dose degasser.

1.2 Mouse Disease Model

The mouse disease models used in this Example were a dilated cardiomyopathy model mouse, a climacterium model mouse resulting from extirpation of the ovary, a sarcoma-transplanted mouse, an Alzheimer's disease model mouse, a DAO deficient mouse and a DDO deficient mouse. Each mouse will be explained in detail below. The experiments in which the mice were used were carried out in Graduate School of Pharmaceutical Sciences, Kyushu University.

1.2.1 Dilated Cardiomyopathy Model Mouse

As the cardiovascular disorder model mouse, MLP-KO mice (Arber, S. et el., Cell, 88: 393 (1997)) that are deficient in MLP (muscle LIM protein) which is one of cardiac muscle constituting proteins were employed. Urine samples from three 8 weeks old male dilated cardiomyopathy model mice (MLP-KO mouse, hereinafter, may be referred to as “diseased”), and four 8 weeks old male control normal mice (hereinafter, may be referred to as “normal”) were obtained, and the total analysis of amino acid optical isomer contents was carried out.

1.2.2 Climacterium Model Mouse

As the climacterium model mouse, the ovary was extirpated from 9 weeks old female HR-1 mice under anesthesia, and the skin was sutured. Also, mice for a control experiment were prepared from the female mice of the same weeks old, through subjecting only to skin incision and suture without subjecting to the extirpation of the ovary. The body weight was measured before the operation, and also one to four weeks after the operation. The individuals whose body weight increase was found to be greater than that of the control mice were subjected to the total analysis of amino acid optical isomer contents in urine as climacterium model mice.

1.2.3 Sarcoma-Transplanted Mouse

As the sarcoma-transplanted mouse, 6 weeks old male ICR mice transplanted with 2×107 sarcoma cells were prepared. Three weeks after the transplantation, the individuals in which growth of the explanted tumor was verified were subjected to the total analysis of amino acid optical isomer contents in urine. As a control experiment of the sarcoma-transplanted mouse, 9 weeks old male ICR mice fed in the same environment were subjected to the total analysis of amino acid optical isomer contents in urine.

1.2.4 Alzheimer's Disease Model Mouse

As the Alzheimer's disease model mouse, 8 weeks old male hemizygote mice of an amyloid β precursor protein-highly expressing transgenic mouse Tg2576 (Hsiao, K. et el., Science, 274: 99 (1996)) were subjected to the total analysis of amino acid optical isomer contents in urine. 8 weeks old male C57BL mice were subjected to the total analysis of amino acid optical isomer contents in urine as control normal mice.

1.2.5 D-Amino Acid Oxidase (DAO) Deficient Mouse

As one of D-amino acid metabolism-related enzyme activity-altered models, 8 weeks old male D-amino acid oxidase (DAO) deficient mice (Konno, R. et el., Genetics 103: 277 (1983), ddY/DAO−, hereinafter, may be referred to as “DAO−/−”) were subjected to the total analysis of amino acid optical isomer contents in urine. As control mice, 8 weeks old male D-amino acid oxidase (DAO) wild type mice were subjected to the total analysis of amino acid optical isomer contents in urine.

1.2.6 D-Aspartate Oxidase (DDO) Deficient Mouse

As one of D-amino acid metabolism-related enzyme activity-altered models, 8 weeks old male D-aspartate oxidase (DDO) deficient mice (Huang, A. S. et el., J. Neurosci., 26: 2814 (2006), hereinafter, may be referred to as “DDO−”) were subjected to the total analysis of amino acid optical isomer contents in urine. As control mice, 8 weeks old male D-aspartate oxidase (DDO) wild type mice (hereinafter, may be referred to as “DDO+”) were subjected to the total analysis of amino acid optical isomer contents in urine.

1.2.7 Phenylketonuria Disease Model Mouse

As one of amino acid metabolic disorder model mice, five 25-35 weeks old male phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mouse, Pah^(enu2)/Pah^(enu2), Shedlovsky, A. et el., Genetics 134: 1205 (1993)) fed under an SPF condition were subjected to the total analysis of amino acid optical isomer contents in urine. As control mice, five 25-35 weeks old male wild type allele homozygote in an identical genetic background, fed under the SPF condition were subjected to the total analysis of amino acid optical isomer contents in urine.

1.2.8 Maple Syrup Urine Disease Model Mouse

As one of amino acid metabolic disorder model mice, five 8-10 weeks old male branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mouse, Dbt^(tmlgeh)/Dbt^(tmlgeh), Homanics G. E. et el., BMC Med Genet, 7: 33 (2006) fed under an SPF condition were subjected to the total analysis of amino acid optical isomer contents in urine. As control mice, five 8-10 weeks old male wild type allele homozygote in an identical genetic background, fed under the SPF condition were subjected to the total analysis of amino acid optical isomer contents in urine. In a part of the experiments, five 8-10 weeks old male +/Dbt^(tmlgeh) heterozygotes fed under the SPF condition as intermediate type mice were subjected to the total analysis of amino acid optical isomer contents in urine.

2. Results

2.1 dilated cardiomyopathy model mouse

FIG. 16 shows a bar chart presenting averages and standard errors of D-serine concentrations in the urine from three dilated cardiomyopathy model mice (MLP-KO mouse, hereinafter, may be referred to as “diseased”) and four control normal mice (hereinafter, may be referred to as “normal”). The ordinate indicates the D-serine concentration (nanomol/mL). FIG. 17 shows a bar chart presenting averages and standard errors of L-serine concentrations in the urine from three dilated cardiomyopathy model mice and four control normal mice. The ordinate indicates the L-serine concentration (nanomol/mL). FIG. 18 shows a bar chart presenting averages and standard errors of total serine concentrations (the sums of D-serine concentration and L-serine concentration) in the urine from three dilated cardiomyopathy model mice and four control normal mice. The ordinate indicates the total serine concentration (nanomol/mL). FIG. 19 shows a bar chart presenting averages and standard errors of the percentages of D-serine concentration (% D) with respect to the total serine concentration in the urine from three dilated cardiomyopathy model mice and four control normal mice. The ordinate indicates the % D. In regard to the significant difference between the normal and the diseased, P was less than 0.02 according to a Student's t-test. FIG. 20 shows a bar chart presenting averages and standard errors of D-arginine concentrations in the urine from three dilated cardiomyopathy model mice and four control normal mice. The ordinate indicates the D-arginine concentration (nanomol/mL). FIG. 21 shows a bar chart presenting averages and standard errors of L-arginine concentrations in the urine from three dilated cardiomyopathy model mice and four control normal mice. The ordinate indicates the L-arginine concentration (nanomol/mL). In regard to the significant difference between the normal and the diseased, P was less than 0.01 according to a Student's t-test. FIG. 22 shows a bar chart presenting averages and standard errors of total arginine concentrations (the sums of D-arginine concentration and L-arginine concentration) in the urine from three dilated cardiomyopathy model mice and four control normal mice. The ordinate indicates the total arginine concentration (nanomol/mL). FIG. 23 shows a bar chart presenting averages and standard errors of D-glutamic acid concentrations in the urine from three dilated cardiomyopathy model mice and four control normal mice. The ordinate indicates the D-glutamic acid concentration (nanomol/mL). In regard to the significant difference between the normal and the diseased, P was less than 0.02 according to a Student's t-test. FIG. 24 shows a bar chart presenting averages and standard errors of the L-glutamic acid concentrations in the urine from three dilated cardiomyopathy model mice and four control normal mice. The ordinate indicates the L-glutamic acid concentration (nanomol/mL). FIG. 25 shows a bar chart presenting averages and standard errors of total glutamic acid concentrations (the sums of D-glutamic acid concentration and L-glutamic acid concentration) in the urine from three dilated cardiomyopathy model mice and four control normal mice. The ordinate indicates the total glutamic acid concentration (nanomol/mL). FIG. 26 shows a bar chart presenting averages and standard errors of D-proline concentrations in the urine from three dilated cardiomyopathy model mice and four control normal mice. The ordinate indicates the D-proline concentration (nanomol/mL). In regard to the significant difference between the normal and the diseased, P was less than 0.01 according to a Student's t-test. FIG. 27 shows a bar chart presenting averages and standard errors of L-proline concentrations in the urine from three dilated cardiomyopathy model mice and four control normal mice. The ordinate indicates the L-proline concentration (nanomol/mL). FIG. 28 shows a bar chart presenting averages and standard errors of total proline concentrations (the sums of D-proline concentration and L-proline concentration) in the urine from three dilated cardiomyopathy model mice and four control normal mice. The ordinate indicates the total proline concentration (nanomol/mL). FIG. 29 shows a bar chart presenting averages and standard errors of D-lysine concentrations in the urine from three dilated cardiomyopathy model mice and four control normal mice. The ordinate indicates the D-lysine concentration (nanomol/mL). In regard to the significant difference between the normal and the diseased, P was less than 0.01 according to a Student's t-test. FIG. 30 shows a bar chart presenting averages and standard errors of L-lysine concentrations in the urine from three dilated cardiomyopathy model mice and four control normal mice. The ordinate indicates the L-lysine concentration (nanomol/mL). FIG. 31 shows a bar chart presenting averages and standard errors of total lysine concentrations (the sums of D-lysine concentration and L-lysine concentration) in the urine from three dilated cardiomyopathy model mice and four control normal mice. From these results, the D-serine concentration tended to be lower in the diseased group than in the normal group, but a significant difference was not found. However, according to evaluations on the basis of “% D”, % D of D-serine was significantly smaller in the diseased group than in the normal group. The L-arginine concentration was significantly lower in the diseased group than in the normal group. The D-glutamic acid concentration was significantly higher in the diseased group than in the normal group. The D-proline concentration was significantly lower in the diseased group than in the normal group. The D-lysine concentration was significantly lower in the diseased group than in the normal group.

2.2 Climacterium Model Mouse

FIG. 32 shows a graph presenting the changes in averages and standard deviations of the body weights of 12 climacterium model mice (hereinafter, may be referred to as “OVX”) obtained by extirpation of the ovary from 9 weeks old HR-1 mice, and six control mice (hereinafter, may be referred to as “sham”) subjected to only skin incision and suture without carrying out the extirpation of the ovary from female mice of the same weeks old. The ordinate indicates the body weight (gram) of the mice. The black solid bars and the diagonally hatched bars show the average and standard deviation of the body weights of: before the operation (9 weeks old) and one week (10 weeks old), two weeks (11 weeks old), three weeks (12 weeks old) and four weeks (13 weeks old) after the operation of both climacterium model mice (OVX) and control mice (sham), respectively. In regard to the significant difference of the body weights between the climacterium model mice (OVX) and the control mice (sham), P was less than 0.05 (*) or less than 0.01 (**) according to a Student's t-test. In climacterium model mice obtained by extirpation of the ovary, the body weight increase was significantly greater than that of the control mice from two weeks after the operation, suggesting that the climacterium model mice were successfully produced.

FIGS. 33, 34 and 35 show Tables presenting results of the total analysis of amino acid optical isomer contents in the urine from the first mouse (OVX-3), the second mouse (OVX-4) and the third mouse (OVX-5), respectively, among three climacterium model mice (OVX). Since no optical isomer of glycine is present, the results on glycine are described in a column of “L-form”. All amino acids were fluorescent-derivatized with NBD-F, and analyzed using the apparatus for the total analysis of amino acid optical isomer contents of the embodiment of the present invention. Since the NBD derivative of tryptophan is poorly sensitive, “nd” was designated in this analysis. Since cysteine produces cystine by air oxidization, the content of cysteine is extremely low, and thus “nd” was designated in this analysis. FIGS. 36, 32, 33 and 34 show Tables presenting results of the total analysis of amino acid optical isomer contents in the urine from the first mouse (Sham-16), the second mouse (Sham-17), the third mouse (Sham-19) and the fourth mouse (Sham-20), respectively, among four control mice (sham). FIG. 40 shows a Table comparing D-amino acid contents in the urine from three climacterium model mice (OVX) and four control mice (sham). The D-aspartic acid content was lower in the control mice (sham) than in the climacterium model mice (OVX), and in regard to the significant difference, P was 0.015 according to a Student's t-test. FIG. 41 shows a Table comparing L-amino acid contents in the urine from three climacterium model mice (OVX) and four control mice (sham). The contents of L-histidine and L-phenylalanine were lower in the climacterium model mice (OVX) than in the control mice (sham), and in regard to the significant difference, P was 0.017 and 0.037, respectively, according to a Student's t-test. FIG. 42 shows a Table comparing percentages of the D-form concentration (% D) with respect to the sum of the D-form concentration and the L-form concentration of each amino acid in the urine from three climacterium model mice (OVX) and four control mice (sham). Since D-forms of threonine and isoleucine turn to be allo-forms in a living body, the % D was evaluated in terms of the percentage of the D-allo-form concentration with respect to the sum of the D-allo-form concentration and the L-form concentration. The percentage of D-aspartic acid was lower in the climacterium model mice (OVX) than in the control mice (sham), and in regard to the significant difference, P was 0.002 according to a Student's t-test. FIG. 43 shows a Table comparing percentages of each D-amino acid concentration (% D/total L) with respect to the sum of the concentrations of all the L-amino acids in the urine from three climacterium model mice (OVX) and four control mice (sham). The percentage of D-aspartic acid was lower in the climacterium model mice (OVX) than in the control mice (sham), and in regard to the significant difference, P was 0.005 according to a Student's t-test. FIG. 44 shows a Table comparing percentages of each D-amino acid concentration (% D/total D) with respect to the sum of the concentrations of all the D-amino acids in the urine from three climacterium model mice (OVX) and four control mice (sham). The percentage of D-aspartic acid was lower in the climacterium model mice (OVX) than in the control mice (sham), and in regard to the significant difference, P was 0.017 according to a Student's t-test. FIG. 45 shows a Table comparing percentages of each D-amino acid concentration with respect to the D-glutamic acid concentration (% D/D-Glu) in the urine from three climacterium model mice (OVX) and four control mice (sham). Since D-aspartic acid is an acidic D-amino acid, an evaluation was made after a correction with the concentration of D-glutamic acid which is considered to be similarly metabolized. Also in the case of % D/D-Glu, The percentage of D-aspartic acid was lower in the climacterium model mice (OVX) than in the control mice (sham), and in regard to the significant difference, P was 0.006 according to a Student's t-test.

2.3 Sarcoma-Transplanted Mouse

FIGS. 46, 47 and 48 show Tables presenting results of the total analysis of amino acid optical isomer contents in the urine from the first mouse (S3), the second mouse (S4) and the third mouse (S5), respectively, among three 6 weeks old male ICR mice transplanted with 2×107 sarcoma cells in which growth of the explanted tumor was verified three weeks after the transplantation. Since no optical isomer of glycine is present, the results on glycine are described in a column of “L-form”. All amino acids were fluorescent-derivatized with NBD-F, and analyzed using the apparatus for the total analysis of amino acid optical isomer contents of the embodiment of the present invention. Since the NBD derivative of tryptophan is poorly sensitive, “nd” was designated in this analysis. Since cysteine produces cystine by air oxidization, the content of cysteine is extremely low, and thus “nd” was designated in this analysis. FIGS. 49, 50 and 51 show Tables presenting results of the total analysis of amino acid optical isomer contents in the urine from the first mouse (C3), the second mouse (C4) and the third mouse (C5), respectively, among three 9 weeks old male ICR mice fed for a control experiment in the same environment as that of the sarcoma-transplanted mice. FIG. 52 shows a Table comparing D-amino acid contents in the urine from three sarcoma-transplanted mice (S3, S4 and S5) and three control mice (C3, C4 and C5). FIG. 53 shows a Table comparing L-amino acid contents in the urine from three sarcoma-transplanted mice (S3, S4 and S5) and three control mice (C3, C4 and C5). FIG. 54 shows a Table comparing percentages of the D-form concentration (% D) with respect to the sum of the D-form concentration and the L-form concentration of each amino acid in the urine from three sarcoma-transplanted mice (S3, S4 and S5) and three control mice (C3, C4 and C5). The % D of serine was lower in the sarcoma-transplanted mice than in the control mice, and in regard to the significant difference, P was 0.016 according to a Student's t-test. FIG. 55 shows a Table comparing percentages of each D-amino acid concentration (% D/total L) with respect to the sum of the concentrations of all the L-amino acids in the urine from three sarcoma-transplanted mice (S3, S4 and S5) and three control mice (C3, C4 and C5). FIG. 56 shows a Table comparing percentages of each D-amino acid concentration (% D/total D) with respect to the sum of the concentrations of all the D-amino acids in the urine from three sarcoma-transplanted mice (S3, S4 and S5) and three control mice (C3, C4 and C5). The percentages of D-asparagine and D-arginine tended to be greater in the sarcoma-transplanted mice than in the control mice, and in regard to the significant difference of D-arginine, P was 0.035 according to a Student's t-test. FIG. 57 shows a Table comparing percentages of each D-amino acid concentration with respect to the D-asparagine concentration (% D/D-Asn) in the urine from three sarcoma-transplanted mice (S3, S4 and S5) and three control mice (C3, C4 and C5). D-asparagine was used as a basis for a correction of the concentration in the urine since D-asparagine is present in mammalian urine at a comparatively high concentration, and the proportion with respect to the total D-amino acid concentrations was most stable. The percentages of D-arginine tended to be greater in the sarcoma-transplanted mice than in the control mice, and in regard to the significant difference, P was 0.016 according to a Student's t-test.

2.4 Alzheimer's Disease Model Mouse

FIG. 58 shows a bar chart presenting averages and standard errors of D-serine concentrations (D-Ser), L-serine concentrations (L-Ser) and the sum of both concentrations (Ser) in the urine from three Alzheimer's disease model mice (8 weeks old male hemizygote mice of an amyloid β precursor protein-highly expressing transgenic mouse Tg2576, hereinafter, may be referred to as “hemi”) and three control normal mice (C57BL, hereinafter, may be referred to as “Wild”). The ordinate indicates the concentration (nanomol/mL). FIG. 59 shows a bar chart presenting averages and standard errors of the percentages of D-serine concentration (% D) with respect to the total serine concentration in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). The ordinate indicates the % D. FIG. 60 shows a graph presenting a relative ratio of the D-serine concentration to the D-allo-threonine concentration (D-Ser/D-allo-Thr) or a relative ratio of the D-serine concentration to the D-allo-isoleucine concentration (D-Ser/D-allo-Ile) in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). Both the relative ratio of the D-serine concentration to the D-allo-threonine concentration in the urine, and the relative ratio of the D-serine concentration to the D-allo-isoleucine concentration in the urine were higher in the Alzheimer's disease model mice than in the control mice, and in regard to the significant difference, P was less than 0.01 according to a Student's t-test in both cases. FIG. 61 shows a bar chart presenting averages and standard errors of D-alanine concentrations (D-Ala), L-alanine concentrations (L-Ala) and the sum of both concentrations (Ala) in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). The ordinate indicates the concentration (nanomol/mL). The D-alanine concentration in the urine was higher in the Alzheimer's disease model mice than in the control normal mice, and in regard to the significant difference, P was less than 0.01 according to a Student's t-test. FIG. 62 shows a bar chart presenting averages and standard errors of the percentages of D-alanine concentration (% D) with respect to the total alanine concentration in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). The ordinate indicates the % D. The percentage of the D-alanine concentration (% D) with respect to the total alanine concentration in the urine was higher in the Alzheimer's disease model mice than in the control normal mice, and in regard to the significant difference, P was less than 0.01 according to a Student's t-test. FIG. 63 shows a bar chart presenting averages and standard errors of D-methionine concentrations (D-Met), L-methionine concentrations (L-Met) and the sum of both concentrations (Met) in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). The ordinate indicates the concentration (nanomol/mL). The D-Met concentration in the urine tended to be higher in the Alzheimer's disease model mice than in the control normal mice. FIG. 64 shows a bar chart presenting averages and standard errors of the percentages of D-methionine concentration (% D) with respect to the total serine concentration in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). The ordinate indicates the % D. FIG. 65 shows a graph presenting a relative ratio of the D-methionine concentration to the D-allo-threonine concentration (D-Met/D-allo-Thr) or a relative ratio of the D-methionine concentration to the D-allo-isoleucine concentration (D-Met/D-allo-Ile) in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). Both the relative ratio of the D-methionine concentration to the D-allo-threonine concentration in the urine, and the relative ratio of the D-methionine concentration to the D-allo-isoleucine concentration in the urine were higher in the Alzheimer's disease model mice than in the control mice, and in regard to the significant difference, P was less than 0.05 according to a Student's t-test in both cases. FIG. 66 shows a bar chart presenting averages and standard errors of D-leucine concentrations (D-Leu), L-leucine concentrations (L-Leu) and the sum of both concentrations (Leu) in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). The ordinate indicates the concentration (nanomol/mL). FIG. 67 shows a bar chart presenting averages and standard errors of the percentages of D-leucine concentration (% D) with respect to the total serine concentration in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). FIG. 68 shows a graph presenting a relative ratio of the D-leucine concentration to the D-allo-threonine concentration (D-Leu/D-allo-Thr) or a relative ratio of the D-leucine concentration to the D-allo-isoleucine concentration (D-Leu/D-allo-Ile) in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). Both the relative ratio of the D-leucine concentration to the D-allo-threonine concentration in the urine, and the relative ratio of the D-leucine concentration to the D-allo-isoleucine concentration in the urine were higher in the Alzheimer's disease model mice than in the control mice, and in regard to the significant difference, P was less than 0.05 and less than 0.01, respectively, according to a Student's t-test. FIG. 69 shows a bar chart presenting averages and standard errors of D-aspartic acid concentrations (D-Asp), L-aspartic acid concentrations (L-Asp) and the sum of both concentrations (Asp) in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). The ordinate indicates the concentration (nanomol/mL). The D-aspartic acid concentration in the urine was lower in the Alzheimer's disease model mice than in the control normal mice, and in regard to the significant difference, P was less than 0.05 according to a Student's t-test. FIG. 70 shows a bar chart presenting averages and standard errors of the percentages of the D-aspartic acid concentration (% D) with respect to the total aspartic acid concentration in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). The ordinate indicates the % D. FIG. 71 shows a bar chart presenting averages and standard errors of D-phenylalanine concentrations (D-Phe), L-phenylalanine concentrations (L-Phe) and the sum of both concentrations (Phe) in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). The ordinate indicates the concentration (nanomol/mL). FIG. 72 shows a bar chart presenting averages and standard errors of the percentages of D-phenylalanine concentration (% D) with respect to the total phenylalanine concentration in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). FIG. 73 shows a graph presenting a relative ratio of the D-phenylalanine concentration to the D-allo-threonine concentration (D-Phe/D-allo-Thr) or a relative ratio of the D-phenylalanine concentration to the D-allo-isoleucine concentration (D-Phe/D-allo-Ile) in the urine from three Alzheimer's disease model mice (hemi) and three control normal mice (Wild). The relative ratio of the D-phenylalanine concentration to the D-allo-isoleucine concentration in the urine was higher in the Alzheimer's disease model mice than in the control mice, and in regard to the significant difference, P was less than 0.05 according to a Student's t-test.

2.5 D-Amino Acid Oxidase (DAO) Deficient Mouse

FIGS. 74, 75 and 76 show Tables presenting results of the total analysis of amino acid optical isomer contents in the urine from the first mouse (DAO+/+1), the second mouse (DAO+/+2) and the third mouse (DAO+/+3), respectively, among three D-amino acid oxidase (DAO) wild type mice (ddY/DAO+, which may be referred to as “DAO+/+”). Since no optical isomer of glycine is present, the results on glycine are described in a column of “L-form”. All amino acids were fluorescent-derivatized with NBD-F, and analyzed using the apparatus for the total analysis of amino acid optical isomer contents of the embodiment of the present invention. Since the NBD derivative of tryptophan is poorly sensitive, “nd” was designated in this analysis. Since cysteine produces cystine by air oxidization, the content of cysteine is extremely low, and thus “nd” was designated in this analysis. FIGS. 77, 78 and 79 show Tables presenting results of the total analysis of amino acid optical isomer contents in the urine from the first mouse (DAO−/−1), the second mouse (DAO−/−2) and the third mouse (DAO−/−3), respectively, among three D-amino acid oxidase (DAO) deficient mice (ddY/DAO−, hereinafter, may be referred to as “DAO−/−”). Since no optical isomer of glycine is present, the results on glycine are described in a column of “L-form”. All amino acids were fluorescent-derivatized with NBD-F, and analyzed using the apparatus for the total analysis of amino acid optical isomer contents of the embodiment of the present invention. Since the NBD derivative of tryptophan is poorly sensitive, “nd” was designated in this analysis. Since cysteine produces cystine by air oxidization, the content of cysteine is extremely low, and thus “nd” was designated in this analysis. FIG. 80 shows a Table comparing D-amino acid contents in the urine from three D-amino acid oxidase (DAO) wild type mice (DAO+/+1, DAO+/+2 and DAO+/+3) and three D-amino acid oxidase (DAO) deficient mice (DAO−/−1, DAO−/−2 and DAO−/−3). The contents of D-serine, D-allo-threonine, D-alanine, D-proline, D-leucine and D-phenylalanine were significantly higher in the D-amino acid oxidase (DAO) enzyme deficient mice than in the DAO wild type mice. FIG. 81 shows a Table comparing L-amino acid contents in the urine from three D-amino acid oxidase (DAO) wild type mice (DAO+/+1, DAO+/+2 and DAO+/+3) and three D-amino acid oxidase (DAO) deficient mice (DAO−/−1, DAO−/−2 and DAO−/−3). The contents of D-serine, D-allo-threonine, D-alanine, D-proline, D-leucine and D-phenylalanine were significantly higher in the D-amino acid oxidase (DAO) enzyme deficient mice than in the DAO wild type mice. FIG. 82 shows a Table comparing percentages of the D-form concentration (% D) with respect to the sum of the D-form concentration and the L-form concentration of each amino acid in the urine from three D-amino acid oxidase (DAO) wild type mice (DAO+/+1, DAO+/+2 and DAO+/+3) and three D-amino acid oxidase (DAO) deficient mice (DAO−/−1, DAO−/−2 and DAO−/−3). The percentages of D-serine, D-allo-threonine, D-alanine, D-proline, D-leucine and D-phenylalanine were significantly higher in the D-amino acid oxidase (DAO) enzyme deficient mice than in the DAO wild type mice. FIG. 83 shows a Table comparing percentages of each D-amino acid concentration (% D/total L) with respect to the sum of the concentrations of all the L-amino acids in the urine from three D-amino acid oxidase (DAO) wild type mice (DAO+/+1, DAO+/+2 and DAO+/+3) and three D-amino acid oxidase (DAO) deficient mice (DAO−/−1, DAO−/−2 and DAO−/−3). The percentages of D-serine, D-allo-threonine, D-alanine, D-leucine and D-phenylalanine were significantly higher in the D-amino acid oxidase (DAO) enzyme deficient mice than in the DAO wild type mice. FIG. 84 shows a Table comparing percentages of each D-amino acid concentration (% D/total D) with respect to the sum of the concentrations of all the D-amino acids in the urine from three D-amino acid oxidase (DAO) wild type mice (DAO+/+1, DAO+/+2 and DAO+/+3) and three D-amino acid oxidase (DAO) deficient mice (DAO−/−1, DAO−/−2 and DAO−/−3). FIG. 85 shows a Table comparing percentages of each D-amino acid concentration (% D/D-Asn) with respect to the D-asparagine concentration in the urine from three D-amino acid oxidase (DAO) wild type mice (DAO+/+1, DAO+/+2 and DAO+/+3) and three D-amino acid oxidase (DAO) deficient mice (DAO−/−1, DAO−/−2 and DAO−/−3). The percentages of D-serine, D-allo-threonine, D-alanine, D-proline, D-leucine and D-phenylalanine were significantly higher in the D-amino acid oxidase (DAO) enzyme deficient mice than in the DAO wild type mice.

2.6 D-Aspartate Oxidase (DDO) Deficient Mouse

FIGS. 86, 87, 88 and 89 show wave form charts presenting results of the total analysis of amino acid optical isomer contents in the urine from the first mouse, the second mouse, the third mouse and the fourth mouse, respectively, among four D-aspartate oxidase (DDO) wild type mice (DDO+). FIGS. 90, 91, 92 and 93 show wave form charts presenting results of the total analysis of amino acid optical isomer contents in the urine from the first mouse, the second mouse, the third mouse and the fourth mouse, respectively, among four D-aspartate oxidase (DDO) deficient mice (DDO−). The D-asparagine concentration was higher in the urine from the DDO deficient mice than in the urine from the wild type mice, and the concentrations of D-aspartic acid and D-arginine were also higher in the urine from the DDO deficient mice. D-glutamic acid which is a good substrate for the DDO enzyme was scarcely found in the urine from the DDO deficient mice.

From the results discussed in 2.5 and 2.6 above, deficiency of DAO and DDO that are D-amino acid metabolic enzymes resulted in change in the concentrations of the D-amino acids specific to these enzymes, respectively. In other words, it is believed that the specific D-amino acid concentrations can be controlled by controlling the activities of these enzymes.

2.7 Phenylketonuria Disease Model Mouse

FIG. 94 shows a bar chart presenting D-valine concentrations (nanomol/mL) and standard errors thereof in the plasma from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid). FIG. 95 shows a bar chart presenting D-allo-isoleucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid). FIG. 96 shows a bar chart presenting D-isoleucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid). FIG. 97 shows a bar chart presenting D-leucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid)_(a)nd five control mice (white solid). FIG. 98 shows a bar chart presenting D-phenylalanine concentrations (nanomol/mL) and standard errors thereof in the plasma from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid). The D-phenylalanine concentration was higher in the phenylketonuria disease model mice than in the control mice, and in regard to the significant difference, p was less than 0.01. FIG. 99 shows a bar chart presenting L-valine concentrations (nanomol/mL) and standard errors thereof in the plasma from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid). FIG. 100 shows a bar chart presenting L-allo-isoleucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid). FIG. 101 shows a bar chart presenting L-isoleucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid). FIG. 102 shows a bar chart presenting L-leucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid). FIG. 103 shows a bar chart presenting L-phenylalanine concentrations (nanomol/mL) and standard errors thereof in the plasma from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid). The concentration of L-phenylalanine was higher in the phenylketonuria disease model mice than in the control mice, and in regard to the significant difference, p was less than 0.01.

FIG. 104 shows a bar chart presenting D-valine concentrations (nanomol/mL) and standard errors thereof in the urine from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid). FIG. 105 shows a bar chart presenting D-allo-isoleucine concentrations (nanomol/mL) and standard errors thereof in the urine from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid). FIG. 106 shows a bar chart presenting D-isoleucine concentrations (nanomol/mL) and standard errors thereof in the urine from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid). FIG. 107 shows a bar chart presenting D-leucine concentrations (nanomol/mL) and standard errors thereof in the urine from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid). FIG. 108 shows a bar chart presenting D-phenylalanine concentrations (nanomol/mL) and standard errors thereof in the urine from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid). FIG. 109 shows a bar chart presenting L-valine concentrations (nanomol/mL) and standard errors thereof in the urine from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid). FIG. 110 shows a bar chart presenting L-allo-isoleucine concentrations (nanomol/mL) and standard errors thereof in the urine from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid). FIG. 111 shows a bar chart presenting L-isoleucine concentrations (nanomol/mL) and standard errors thereof in the urine from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid). FIG. 112 shows a bar chart presenting L-leucine concentrations (nanomol/mL) and standard errors thereof in the urine from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid) and five control mice (white solid). FIG. 113 shows a bar chart presenting L-phenylalanine concentrations (nanomol/mL) and standard errors thereof in the urine from five phenylketonuria disease model mice (phenylalanine hydroxylase (PAH) mutant mice, Pah^(enu2)/Pah^(enu2); black solid)_(a)nd five control mice (white solid). The L-phenylalanine concentration was higher in the phenylketonuria disease model mice than in the control mice, and in regard to the significant difference, p was less than 0.01.

2.8 Maple Syrup Urine Disease Model Mouse

FIG. 114 shows a bar chart presenting D-valine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). FIG. 115 shows a bar chart presenting D-allo-isoleucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). FIG. 116 shows a bar chart presenting D-isoleucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). The D-isoleucine concentration in the plasma was higher in the maple syrup urine disease mice than in the control mice, and in regard to the significant difference, p was less than 0.05. FIG. 117 shows a bar chart presenting D-leucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). FIG. 118 shows a bar chart presenting D-phenylalanine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). FIG. 119 shows a bar chart presenting L-valine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). The L-valine concentration in the plasma was higher in the maple syrup urine disease mice than in the control mice, and in regard to the significant difference, p is less than 0.01. FIG. 120 shows a bar chart presenting L-allo-isoleucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). The L-allo-isoleucine concentration in the plasma was higher in the maple syrup urine disease mice than in the control mice, and in regard to the significant difference, p was less than 0.01. FIG. 121 shows a bar chart presenting L-isoleucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). The L-isoleucine concentration in the plasma was higher in the maple syrup urine disease mice than in the control mice, and in regard to the significant difference, p was less than 0.01. FIG. 122 shows a bar chart presenting L-leucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). The L-leucine concentration in the plasma was higher in the maple syrup urine disease mice than in the control mice, and in regard to the significant difference, p was less than 0.05. FIG. 123 shows a bar chart presenting L-phenylalanine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). FIG. 124 shows a bar chart presenting D-isoleucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease intermediate type mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, +/Dbt^(tmlgeh); grey solid) and five control mice (white solid). The D-isoleucine concentration in the plasma was higher in the maple syrup urine disease mice than in the control mice, and in regard to the significant difference, p was less than 0.01. FIG. 125 shows a bar chart presenting L-valine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease intermediate type mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, +/Dbt^(tmlgeh); grey solid) and five control mice (white solid). FIG. 126 shows a bar chart presenting L-allo-isoleucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease intermediate type mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, +/Dbt^(tmlgeh); grey solid) and five control mice (white solid). FIG. 127 shows a bar chart presenting L-isoleucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease intermediate type mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, +/Dbt^(tmlgeh); grey solid) and five control mice (white solid). FIG. 128 shows a bar chart presenting L-leucine concentrations (nanomol/mL) and standard errors thereof in the plasma from five maple syrup urine disease intermediate type mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, +/Dbt^(tmlgeh); grey solid) and five control mice (white solid).

FIG. 129 shows a bar chart presenting D-valine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). FIG. 130 shows a bar chart presenting D-allo-isoleucine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). FIG. 131 shows a bar chart presenting D-isoleucine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). The D-isoleucine concentration in the urine was higher in the maple syrup urine disease mice than in the control mice, and in regard to the significant difference, p was less than 0.05. FIG. 132 shows a bar chart presenting D-leucine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). FIG. 133 shows a bar chart presenting D-phenylalanine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). FIG. 134 shows a bar chart presenting L-valine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). The L-valine concentration in the urine was higher in the maple syrup urine disease mouse than in the control mouse, and in regard to the significant difference, p was less than 0.01. FIG. 135 shows a bar chart presenting L-allo-isoleucine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). The L-allo-isoleucine concentration in the urine was higher in the maple syrup urine disease mouse than in the control mouse, and in regard to the significant difference, p was less than 0.01. FIG. 136 shows a bar chart presenting L-isoleucine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). The L-isoleucine concentration in the urine was higher in the maple syrup urine disease mouse than in the control mouse, and in regard to the significant difference, p was less than 0.05. FIG. 137 shows a bar chart presenting L-leucine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). The L-leucine concentration in the urine was higher in the maple syrup urine disease mouse than in the control mouse, and in regard to the significant difference, p was less than 0.01. FIG. 138 shows a bar chart presenting L-phenylalanine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, Dbt^(tmlgeh)/Dbt^(tmlgeh); black solid) and five control mice (white solid). FIG. 139 shows a bar chart presenting D-isoleucine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease intermediate type mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, +/Dbt^(tmlgeh); grey solid) and five control mice (white solid). The D-isoleucine concentration in the urine was higher in the maple syrup urine disease intermediate type mice than in the control mice, and in regard to the significant difference, p was less than 0.05. FIG. 140 shows a bar chart presenting L-valine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease intermediate type mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, +/Dbt^(tmlgeh); grey solid) and five control mice (white solid). FIG. 141 shows a bar chart presenting L-allo-isoleucine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease intermediate type mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, +/Dbt^(tmlgeh); grey solid) and five control mice (white solid). FIG. 142 shows a bar chart presenting L-isoleucine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease intermediate type mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, +/Dbt^(tmlgeh); grey solid) and five control mice (white solid). FIG. 143 shows a bar chart presenting L-leucine concentrations (nanomol/mL) and standard errors thereof in the urine from five maple syrup urine disease intermediate type mice (branched-chain alpha-keto acid dehydrogenase (BCKDH) mutant mice, +/Dbt^(tmlgeh); grey solid) and five control mice (white solid).

Example 3

Total Analysis of Amino Acid Stereoisomers in Samples Derived from Various Types of Disease Patients

(2)

1. Materials and Methods

(1) Samples

Similarly to Example 1, serum samples derived from various types of disease patients were obtained from BioServe Biotechnologies, Ltd., USA, Maryland, Beltsville). The samples were collected by Genomic Collaborative Inc., which was purchased by BioServe Biotechnologies, Ltd. Blood collection was carried out under a control by an attending physician of the patient, and a consent by the blood donor on a document in accordance with United States Health Insurance Portability and Accountability Act (HIPAA) was confirmed. An articular rheumatism sample derived from a 49 years old Caucasian male in a some movable disease state. A kidney cancer sample derived from a 47 years old Vietnamese male falling under stage I. A lung cancer sample derived from a 65 years old Vietnamese male falling under stage I. A cardiovascular disease sample derived from a 43 years old Caucasian male. A multiple sclerosis sample derived from a 33 years old Caucasian male of relapsing-remitting type. An acute myeloid leukemia sample derived from a 16 years old male. A lymphoma sample derived from a 49 years old Vietnamese female. A psoriasis sample derived from a 40 years old Caucasian male. A diabetes sample derived from a 50 years old Caucasian male. A systemic lupus erythematosus sample derived from a 38 years old Caucasian female.

(2) Total Analysis of Amino Acid Stereoisomers

The total analysis of amino acid stereoisomers of the samples was carried out in a similar manner to Example 1.

2. Results

Among the analysis results on disease samples in Example 3, the analysis results of D-amino acids are summarized in FIG. 144, and the analysis results of L-amino acids are summarized in FIG. 145. Similarly to Example 1, each line in the Table shows a disease name of the sample donor, and each row shows the type of D-amino acid analyzed. In Table, ND denotes “being the detection limit or below”. Upward arrowheads indicate that the sample contained the amino acid in an amount more than that in the samples from healthy males shown in the first line, whereas downward arrowheads indicate that the sample contained the amino acid in an amount less than that in the samples from the healthy males shown in the first line. Fine backward diagonal hatching patterns indicate that a ratio of the amount of the amino acid in these samples to the amount of the amino acid in the samples from the healthy males is two times or greater (upward arrowhead), or ½ or less (downward arrowhead). Coarse downward diagonal hatching patterns indicate that a ratio of the amount of the amino acid in these samples to the amount of the amino acid in the samples from the healthy males is less than two times (upward arrowhead), or less than ½ (downward arrowhead). In articular rheumatism, the serum concentration of L-glutamic acid was higher than those in control samples from healthy males, whereas the serum concentrations of L-glutamine and L-cysteine were lower. In kidney cancer, the serum concentration of D-serine was higher than those in the samples from healthy males, whereas the serum concentration of D-alanine was lower. In lung cancer, the serum concentration of D-alanine was lower than those in the samples from healthy males. In cardiovascular disease, the serum concentration of L-arginine was lower than those in the samples from healthy males, whereas the serum concentration of L-glutamic acid was higher. In multiple sclerosis, the serum concentration of D-serine was higher than those in the samples from healthy males, whereas the serum concentration of L-cysteine was lower. In acute myeloid leukemia, the serum concentration of L-cysteine was lower than those in the samples from healthy males. In lymphoma, the serum concentration of L-cysteine was lower than those in the samples from healthy males. In acute lymphocytic leukemia, the serum concentration of L-glutamic acid was higher than those in the samples from healthy males, whereas the L-cysteine concentration was lower. In psoriasis, the serum concentrations of L-arginine and of L-cysteine were lower than those in the samples from healthy males. In diabetes, the serum concentrations of D-alanine and L-cysteine were lower than those in the samples from healthy males, whereas the serum concentration of L-glutamic acid was higher. Note that in systemic lupus erythematosus, no change was found.

Quantitative determination of each amino acid has heretofore been made in terms of the sum of all stereoisomers thereof because of the impossibility of discriminating stereoisomers in accordance with conventional quantitative determination analyses of amino acids. However, according to the diagnostic method of the embodiment of the present invention, stereoisomers can be discriminated as each distinct substance, and a quantitative determination thereof is enabled. As shown in FIG. 13 to FIG. 15, when the amount of the D-form is expressed in terms of the percentage (% D) with respect to the sum of the amounts of the D-form and L-form, less variance was found for the % D of serine in the large intestine cancer patients (FIG. 13) as compared with the D-serine amounts in the large intestine cancer patients (FIG. 7). Similarly, less variance was found for the % D of alanine in the renal disease patients (FIG. 15) as compared with the D-alanine amounts in renal disease patients (FIG. 11). The reason for this phenomenon is assumed that since the amount of the D-amino acid correlates with the disease, and may be able to also correlate with the L-form amount through racemization, it is possible to exclude influences from a change of the L-form amounts, by normalization with the sum of the D-form and L-form. As suggested above, there is a case in which the amount of a D-amino acid correlating with a disease also correlates with the amount of other substance. In this respect, when the amount of the other substance per se does not correlate with the aforementioned disease, a diagnostic characteristics can be further improved by producing a parameter (for example, % D) that normalizes the amount of the D-amino acid with the amount of the other substance.

In near future, investigations of correlations of a large number of diseases with the amount of D-amino acids would be extensively evolved. Accordingly, there exists an expectation for developments of diagnostic techniques for a still larger number of diseases on the basis of the amount of the D-amino acid.

Additional Embodiments

1. An apparatus for analyzing a disease sample comprising: a member for separating and quantitatively determining an amino acid stereoisomer in a biological material from a subject; a member for obtaining an disease state index value through a calculation by substituting an amount of the amino acid stereoisomer into a discriminant equation; and a member for outputting disease state information on the subject on a basis of the disease state index value.

2. The apparatus for analyzing a disease sample according to embodiment 1, wherein the discriminant equation is either: disease state index value=(a measurement value for an amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)/(a reference value for the amino acid stereoisomer that correlates with the disease in a biological material from a healthy individual); or disease state index value=[(a measurement value for an amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)/{(a measurement value for an amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)+(a measurement value for an enantiomer of the amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)}]/[(a reference value for the amino acid stereoisomer that correlates with the disease from among reference values from a healthy individual)/{(a reference value for the amino acid stereoisomer that correlates with the disease from among reference values from the healthy individual)+(a reference value for the enantiomer of the amino acid stereoisomer that correlates with the disease from among reference values from the healthy individual)}].

3. The apparatus for analyzing a disease sample according to embodiment 2, wherein the member for outputting disease state information on the subject on a basis of the disease state index value is a member for outputting disease state information on the subject that the subject is suffering from the disease when the disease state index value is 2.0 or greater.

4. The apparatus for analyzing a disease sample according to embodiment 2, wherein the amino acid stereoisomer that correlates with the disease is: one, or two or more types of amino acids selected from the group consisting of D-serine, D-threonine, D-alanine, D-asparagine, allo-D-threonine, D-glutamine, D-proline and D-phenylalanine when the disease is a renal disease; D-histidine and/or D-asparagine when the disease is prostate gland cancer; D-asparagine when the disease is osteoporosis; D-serine, L-arginine, D-glutamic acid and D-proline when the disease is dilated cardiomyopathy; L-histidine, L-phenylalanine and D-aspartic acid when the disease is a climacteric disorder; D-arginine when the disease is sarcoma; D-allo-isoleucine, D-serine, D-alanine, D-methionine, D-leucine, D-aspartic acid, D-phenylalanine and L-phenylalanine when the disease is Alzheimer's disease; D-serine, D-allo-threonine, D-alanine, D-proline, D-leucine and D-phenylalanine when the disease is DAO deficiency; D-asparagine, D-aspartic acid and D-arginine when the disease is DDO deficiency; L-phenylalanine when the disease is phenylketonuria; L-valine, L-allo-isoleucine, D-isoleucine, L-isoleucine and L-leucine when the disease is a maple syrup urine disease; L-glutamic acid, L-glutamine and L-cysteine when the disease is articular rheumatism; D-serine and D-alanine when the disease is kidney cancer; D-alanine when the disease is lung cancer; L-arginine and L-glutamic acid when the disease is a cardiovascular disease; D-serine and L-cysteine when the disease is multiple sclerosis; L-cysteine when the disease is acute myeloid leukemia; L-cysteine when the disease is lymphoma; L-glutamic acid and L-cysteine when the disease is acute lymphocytic leukemia; L-arginine and L-cysteine when the disease is psoriasis; or D-alanine, L-cysteine and L-glutamic acid when the disease is diabetes.

5. A system for analyzing a disease sample comprising: a quantitative determination analysis unit for separating and quantitatively determining an amino acid stereoisomer in a biological material from a subject; a disease state index value computation unit for obtaining a disease state index value through a calculation by substituting the amount of the amino acid stereoisomer into a discriminant equation; and a disease state information output unit for outputting disease state information on the subject on a basis of the disease state index value.

6. The system for analyzing a disease sample according to embodiment 5, wherein the discriminant equation is either: disease state index value=(a measurement value for an amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)/(a reference value for the amino acid stereoisomer that correlates with the disease in a biological material from a healthy individual); or disease state index value=[(a measurement value for an amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)/{(a measurement value for an amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)+(a measurement value for an enantiomer of the amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)}]/[(a reference value for the amino acid stereoisomer that correlates with the disease from among reference values from a healthy individual)/{(a reference value for the amino acid stereoisomer that correlates with the disease from among reference values from the healthy individual)+(a reference value for the enantiomer of the amino acid stereoisomer that correlates with the disease from among reference values from the healthy individual)}].

7. The system for analyzing a disease sample according to embodiment 6, wherein the disease state information output unit is for outputting disease state information on the subject that the subject is suffering from the disease when the disease state index value is 2.0 or greater.

8. The system for analyzing a disease sample according to embodiment 6, wherein the amino acid stereoisomer that correlates with the disease is: one, or two or more types of amino acids selected from the group consisting of D-serine, D-threonine, D-alanine, D-asparagine, allo-D-threonine, D-glutamine, D-proline and D-phenylalanine when the disease is a renal disease; D-histidine and/or D-asparagine when the disease is prostate gland cancer; D-asparagine when the disease is osteoporosis; D-serine, L-arginine, D-glutamic acid and D-proline when the disease is dilated cardiomyopathy; L-histidine, L-phenylalanine and D-aspartic acid when the disease is a climacteric disorder; D-arginine when the disease is sarcoma; D-allo-isoleucine, D-serine, D-alanine, D-methionine, D-leucine, D-aspartic acid, D-phenylalanine and L-phenylalanine when the disease is Alzheimer's disease; D-serine, D-allo-threonine, D-alanine, D-proline, D-leucine and D-phenylalanine when the disease is DAO deficiency; D-asparagine, D-aspartic acid and D-arginine when the disease is DDO deficiency; L-phenylalanine when the disease is phenylketonuria; L-valine, L-allo-isoleucine, D-isoleucine, L-isoleucine and L-leucine when the disease is a maple syrup urine disease; L-glutamic acid, L-glutamine and L-cysteine when the disease is articular rheumatism; D-serine and D-alanine when the disease is kidney cancer; D-alanine when the disease is lung cancer; L-arginine and L-glutamic acid when the disease is a cardiovascular disease; D-serine and L-cysteine when the disease is multiple sclerosis; L-cysteine when the disease is acute myeloid leukemia; L-cysteine when the disease is lymphoma; L-glutamic acid and L-cysteine when the disease is acute lymphocytic leukemia; L-arginine and L-cysteine when the disease is psoriasis; or D-alanine, L-cysteine and L-glutamic acid when the disease is diabetes.

9. A method for analyzing a disease sample comprising: a step of measuring the amount of an amino acid stereoisomer in a biological material from a subject; a step of obtaining a disease state index value through a calculation by substituting the amount of the amino acid stereoisomer into a discriminant equation; and a step of outputting disease state information on the subject on a basis of the disease state index value.

10. The method for analyzing a disease sample according to embodiment 9, wherein the discriminant equation is either: disease state index value=(a measurement value for an amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)/(a reference value for the amino acid stereoisomer that correlates with the disease in a biological material from a healthy individual); or disease state index value=[(a measurement value for an amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)/{(a measurement value for an amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)+(a measurement value for an enantiomer of the amino acid stereoisomer that correlates with the disease from among the measurement values in a biological material from the subject)}]/[(a reference value for the amino acid stereoisomer that correlates with the disease from among reference values from a healthy individual)/{(a reference value for the amino acid stereoisomer that correlates with the disease from among reference values from the healthy individual)+(a reference value for the enantiomer of the amino acid stereoisomer that correlates with the disease from among reference values from the healthy individual)}].

11. The method for analyzing a disease sample according to embodiment 10, wherein the step of outputting disease state information on the subject on a basis of the disease state index value is a step of outputting disease state information on the subject that the subject is suffering from the disease when the disease state index value is 2.0 or greater.

12. The method for analyzing a disease sample according to embodiment 10, wherein the amino acid stereoisomer that correlates with the disease is: one, or two or more types of amino acids selected from the group consisting of D-serine, D-threonine, D-alanine, D-asparagine, allo-D-threonine, D-glutamine, D-proline and D-phenylalanine when the disease is a renal disease; D-histidine and/or D-asparagine when the disease is prostate gland cancer; D-asparagine when the disease is osteoporosis; D-serine, L-arginine, D-glutamic acid and D-proline when the disease is dilated cardiomyopathy; L-histidine, L-phenylalanine and D-aspartic acid when the disease is a climacteric disorder; D-arginine when the disease is sarcoma; D-allo-isoleucine, D-serine, D-alanine, D-methionine, D-leucine, D-aspartic acid, D-phenylalanine and L-phenylalanine when the disease is Alzheimer's disease; D-serine, D-allo-threonine, D-alanine, D-proline, D-leucine and D-phenylalanine when the disease is DAO deficiency; D-asparagine, D-aspartic acid and D-arginine when the disease is DDO deficiency; L-phenylalanine when the disease is phenylketonuria; L-valine, L-allo-isoleucine, D-isoleucine, L-isoleucine and L-leucine when the disease is a maple syrup urine disease; L-glutamic acid, L-glutamine and L-cysteine when the disease is articular rheumatism; D-serine and D-alanine when the disease is kidney cancer; D-alanine when the disease is lung cancer; L-arginine and L-glutamic acid when the disease is a cardiovascular disease; D-serine and L-cysteine when the disease is multiple sclerosis; L-cysteine when the disease is acute myeloid leukemia; L-cysteine when the disease is lymphoma; L-glutamic acid and L-cysteine when the disease is acute lymphocytic leukemia; L-arginine and L-cysteine when the disease is psoriasis; or D-alanine, L-cysteine and L-glutamic acid when the disease is diabetes.

13. The apparatus for analyzing a disease sample according to embodiment 3, wherein the amino acid stereoisomer that correlates with the disease is: one, or two or more types of amino acids selected from the group consisting of D-serine, D-threonine, D-alanine, D-asparagine, allo-D-threonine, D-glutamine, D-proline and D-phenylalanine when the disease is a renal disease; D-histidine and/or D-asparagine when the disease is prostate gland cancer; D-asparagine when the disease is osteoporosis; D-serine, L-arginine, D-glutamic acid and D-proline when the disease is dilated cardiomyopathy; L-histidine, L-phenylalanine and D-aspartic acid when the disease is a climacteric disorder; D-arginine when the disease is sarcoma; D-allo-isoleucine, D-serine, D-alanine, D-methionine, D-leucine, D-aspartic acid, D-phenylalanine and L-phenylalanine when the disease is Alzheimer's disease; D-serine, D-allo-threonine, D-alanine, D-proline, D-leucine and D-phenylalanine when the disease is DAO deficiency; D-asparagine, D-aspartic acid and D-arginine when the disease is DDO deficiency; L-phenylalanine when the disease is phenylketonuria; L-valine, L-allo-isoleucine, D-isoleucine, L-isoleucine and L-leucine when the disease is a maple syrup urine disease; L-glutamic acid, L-glutamine and L-cysteine when the disease is articular rheumatism; D-serine and D-alanine when the disease is kidney cancer; D-alanine when the disease is lung cancer; L-arginine and L-glutamic acid when the disease is a cardiovascular disease; D-serine and L-cysteine when the disease is multiple sclerosis; L-cysteine when the disease is acute myeloid leukemia; L-cysteine when the disease is lymphoma; L-glutamic acid and L-cysteine when the disease is acute lymphocytic leukemia; L-arginine and L-cysteine when the disease is psoriasis; or D-alanine, L-cysteine and L-glutamic acid when the disease is diabetes.

14. The system for analyzing a disease sample according to embodiment 7, wherein the amino acid stereoisomer that correlates with the disease is: one, or two or more types of amino acids selected from the group consisting of D-serine, D-threonine, D-alanine, D-asparagine, allo-D-threonine, D-glutamine, D-proline and D-phenylalanine when the disease is a renal disease; D-histidine and/or D-asparagine when the disease is prostate gland cancer; D-asparagine when the disease is osteoporosis; D-serine, L-arginine, D-glutamic acid and D-proline when the disease is dilated cardiomyopathy; L-histidine, L-phenylalanine and D-aspartic acid when the disease is a climacteric disorder; D-arginine when the disease is sarcoma; D-allo-isoleucine, D-serine, D-alanine, D-methionine, D-leucine, D-aspartic acid, D-phenylalanine and L-phenylalanine when the disease is Alzheimer's disease; D-serine, D-allo-threonine, D-alanine, D-proline, D-leucine and D-phenylalanine when the disease is DAO deficiency; D-asparagine, D-aspartic acid and D-arginine when the disease is DDO deficiency; L-phenylalanine when the disease is phenylketonuria; L-valine, L-allo-isoleucine, D-isoleucine, L-isoleucine and L-leucine when the disease is a maple syrup urine disease; L-glutamic acid, L-glutamine and L-cysteine when the disease is articular rheumatism; D-serine and D-alanine when the disease is kidney cancer; D-alanine when the disease is lung cancer; L-arginine and L-glutamic acid when the disease is a cardiovascular disease; D-serine and L-cysteine when the disease is multiple sclerosis; L-cysteine when the disease is acute myeloid leukemia; L-cysteine when the disease is lymphoma; L-glutamic acid and L-cysteine when the disease is acute lymphocytic leukemia; L-arginine and L-cysteine when the disease is psoriasis; or D-alanine, L-cysteine and L-glutamic acid when the disease is diabetes.

15. The method for analyzing a disease sample according to embodiment 11, wherein the amino acid stereoisomer that correlates with the disease is: one, or two or more types of amino acids selected from the group consisting of D-serine, D-threonine, D-alanine, D-asparagine, allo-D-threonine, D-glutamine, D-proline and D-phenylalanine when the disease is a renal disease; D-histidine and/or D-asparagine when the disease is prostate gland cancer; D-asparagine when the disease is osteoporosis; D-serine, L-arginine, D-glutamic acid and D-proline when the disease is dilated cardiomyopathy; L-histidine, L-phenylalanine and D-aspartic acid when the disease is a climacteric disorder; D-arginine when the disease is sarcoma; D-allo-isoleucine, D-serine, D-alanine, D-methionine, D-leucine, D-aspartic acid, D-phenylalanine and L-phenylalanine when the disease is Alzheimer's disease; D-serine, D-allo-threonine, D-alanine, D-proline, D-leucine and D-phenylalanine when the disease is DAO deficiency; D-asparagine, D-aspartic acid and D-arginine when the disease is DDO deficiency; L-phenylalanine when the disease is phenylketonuria; L-valine, L-allo-isoleucine, D-isoleucine, L-isoleucine and L-leucine when the disease is a maple syrup urine disease; L-glutamic acid, L-glutamine and L-cysteine when the disease is articular rheumatism; D-serine and D-alanine when the disease is kidney cancer; D-alanine when the disease is lung cancer; L-arginine and L-glutamic acid when the disease is a cardiovascular disease; D-serine and L-cysteine when the disease is multiple sclerosis; L-cysteine when the disease is acute myeloid leukemia; L-cysteine when the disease is lymphoma; L-glutamic acid and L-cysteine when the disease is acute lymphocytic leukemia; L-arginine and L-cysteine when the disease is psoriasis; or D-alanine, L-cysteine and L-glutamic acid when the disease is diabetes. 

1.-13. (canceled)
 14. A method for diagnosing and treating a renal disease of a subject comprising: measuring an amount of an amino acid stereoisomer in a blood sample from a subject; determining whether the subject has a renal disease is suspected of having an early stage of the renal disease based on the measured value of the amount of the amino acid stereoisomer and a reference value from a healthy individual, wherein the amino acid stereoisomer is selected from the group consisting of D-serine, D-threonine, D-alanine, D-asparagine, D-allo-threonine, D-glutamine, D-proline and D-phenylalanine, and treating the renal disease in the subject who is determined to have renal disease or is suspected of having an early stage of renal disease, wherein said treating comprises drug administration, artificial dialysis or kidney transplantation, wherein said drug is a renal disease therapeutic drug that controls the progression of the renal disease or improves the renal disease which is at least one selected from the group consisting of antihypertensive drugs, antidiabetic drugs, antidyslipidemic drugs, antianemic drugs, therapeutic drugs for bone and mineral metabolic disorders, therapeutic drugs for hyperuricemia, and therapeutic drugs for uremic toxins.
 15. The method of claim 14, wherein the drug is at least one selected from the group consisting of angiotensin-converting enzymes, angiotensin II receptor antagonists, α-glucosidase inhibitors, insulin preparations, HMG-CoA reductase inhibitors, intestinal cholesterol transporter inhibitors, recombinant human erythropoietin preparations and spherical adsorbent carbon medications.
 16. The method of claim 14, wherein said blood sample is a serum sample.
 17. The method of claim 15, wherein said blood sample is a serum sample.
 18. The method of claim 14, wherein the drug is selected from a therapeutic drug for hyperkalemia and a therapeutic drug for hyperphosphatemia.
 19. The method of claim 15, wherein the drug is selected from a therapeutic drug for hyperkalemia and a therapeutic drug for hyperphosphatemia.
 20. The method of claim 14, further comprising calculating a disease state index by substituting the measured amount of the amino acid stereoisomer into a discriminant equation; and wherein said determining comprises determining that the subject has the renal disease or is suspected of having an early stage of the renal disease if the calculated value of the disease state index is statistically different compared to a healthy individual group disease state index reference value.
 21. The method of claim 15, further comprising calculating a disease state index by substituting the measured amount of the amino acid stereoisomer into a discriminant equation; and wherein said determining comprises determining that the subject has the renal disease or is suspected of having an early stage of the renal disease if the calculated value of the disease state index is statistically different compared to a healthy individual group disease state index reference value.
 22. The method of claim 20, wherein the determining step comprises a) the subject has renal disease if the calculated value of the disease state index for the subject is statistically similar to a healthy individual group disease state index reference value; b) the subject has renal disease if the calculated value of the disease state index for the subject is statistically similar to an acute or chronic renal disease patient disease state index reference value; and c) the subject is suspected of having an early stage of renal disease if the calculated value of the disease state index for the subject is between a healthy individual group disease state index reference value and an acute or chronic renal disease patient disease state index reference value.
 23. The method of claim 21, wherein the determining step comprises a) the subject has renal disease if the calculated value of the disease state index for the subject is statistically similar to a healthy individual group disease state index reference value; b) the subject has renal disease if the calculated value of the disease state index for the subject is statistically similar to an acute or chronic renal disease patient disease state index reference value; and c) the subject is suspected of having an early stage of renal disease if the calculated value of the disease state index for the subject is between a healthy individual group disease state index reference value and an acute or chronic renal disease patient disease state index reference value.
 24. The method of claim 20, wherein when the subject is determined to be suspected of having an early stage of renal disease, the renal disease is improved or the progression of renal disease is controlled, before the subject exhibits blood creatinine level fluctuations.
 25. The method of claim 21, wherein when the subject is determined to be suspected of having an early stage of renal disease, the renal disease is improved or the progression of renal disease is controlled, before the subject exhibits blood creatinine level fluctuations
 26. The method of claim 20, which does not comprise measuring a volume of the blood sample and wherein said calculating does not comprise correcting for the volume of the blood sample.
 27. The method of claim 21, which does not comprise measuring a volume of the blood sample and wherein said calculating does not comprise correcting for the volume of the blood sample.
 28. The method of claim 20, wherein said calculating comprises calculating a ratio between the measured concentration of D-serine and the measured concentration of L-serine or calculating a ratio between the measured concentration of D-serine and a sum of the measured concentration of D-serine and the measured concentration of L-serine.
 29. The method of claim 21, wherein said calculating comprises calculating a ratio between the measured concentration of D-serine and the measured concentration of L-serine or calculating a ratio between the measured concentration of D-serine and a sum of the measured concentration of D-serine and the measured concentration of L-serine.
 30. The method of claim 20, further comprising measuring a pair of concentrations of D-form and L-form of amino acids other than serine, using a pathological index calculated from the pair of concentrations in combination with said pathological index to determine that the subject has renal disease or is suspected of having an early stage of renal disease.
 31. The method of claim 21, further comprising measuring a pair of concentrations of D-form and L-form of amino acids other than serine, using a pathological index calculated from the pair of concentrations in combination with said pathological index to determine that the subject has renal disease or is suspected of having an early stage of renal disease.
 32. The method of claim 14, wherein said measuring is performed by means of a separation analysis system comprising a chiral column.
 33. The method of claim 32, wherein the separation analysis system is a HPLC system. 